Figure 2 ( A ) TOM20 - positive MDV formation was induced in SH - SY5Y by siRNA knockdown of Tollip . MDVs were defined as TOM20 + ve / Cytochrome c ( Cyt c ) - ve via immunofluorescence microscopy , which indicated some colocalisation with Parkin ( arrowheads ) . ( B ) SH - SY5Y cells were transfected with GFP - Tollip for 24 hours , then treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or AO / 100 μM bafilomycin A1 ( BfnA1 ) for 2 hours prior to fixation and immunostaining . Antibodies specific to GFP ( green ) , Parkin ( red ) and ubiquitin ( blue ) were used . C ) GFP - Tollip / Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO / BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell , across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ) . Results are represented as a percentage of Tollip puncta positive for Parkin . ( D ) SH - SY5Y cells expressing GFP - Tollip were immunostained for GFP ( green ) , Parkin ( red ) , and Rab7a ( blue ) . Arrowheads indicate areas of colocalisation between all three markers . ( E - F ) SH - SY5Y cells were transfected with GFP - Tollip R78A or CUE mutant for 24 hours , then treated with AO or AO / BfnA1 for 2 hours prior to fixation and immunostaining ( G ) The percentage of GFP - Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0162 , * * p = 0 . 0034 , * * * * p < 0 . 0001 ) . ( H ) The percentage of ubiquitin puncta positive for Tollip was quantitated from immunuofluorescence images immunostained for ubiquitin and GFP from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0382 , * * p = 0 . 0068 ) . ( I ) The percentage of ubiquitin puncta positive for Parkin was quantitated from immunuofluorescence images immunostained for ubiquitin and Parkin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0232 , * * p = 0 . 0062 ) . ( J ) The percentage of GFP - Tollip / Parkin vesicles positive for ubiquitin were quantitated from immunofluorescence images immunostained for GFP , Parkin , and ubiquitin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * * p = 0 . 0070 ) .

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

Figure 2(A) TOM20-positive MDV formation was induced in SH-SY5Y by siRNA knockdown of Tollip. MDVs were defined as TOM20+ve/Cytochrome c (Cyt c)-ve via immunofluorescence microscopy, which indicated some colocalisation with Parkin (arrowheads).(B) SH-SY5Y cells were transfected with GFP-Tollip for 24 hours, then treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A1 (BfnA1) for 2 hours prior to fixation and immunostaining. Antibodies specific to GFP (green), Parkin (red) and ubiquitin (blue) were used.C) GFP-Tollip/Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO/BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell, across 3-6 GFP-transfected cells/condition, per experiment (n = 3). Results are represented as a percentage of Tollip puncta positive for Parkin.(D) SH-SY5Y cells expressing GFP-Tollip were immunostained for GFP (green), Parkin (red), and Rab7a (blue). Arrowheads indicate areas of colocalisation between all three markers.(E-F) SH-SY5Y cells were transfected with GFP-Tollip R78A or CUE mutant for 24 hours, then treated with AO or AO/BfnA1 for 2 hours prior to fixation and immunostaining(G) The percentage of GFP-Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/ condition, per experiment (n = 3; *p=0.0162, **p=0.0034, ****p<0.0001).(H) The percentage of ubiquitin puncta positive for Tollip was quantitated from immunuofluorescence images immunostained for ubiquitin and GFP from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0382, **p=0.0068).(I) The percentage of ubiquitin puncta positive for Parkin was quantitated from immunuofluorescence images immunostained for ubiquitin and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0232, **p=0.0062).(J) The percentage of GFP-Tollip/Parkin vesicles positive for ubiquitin were quantitated from immunofluorescence images immunostained for GFP, Parkin, and ubiquitin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; **p=0.0070).


Figure 3 ( A ) HeLa cells expressing mycBioID - Tollip and HA - Parkin were either left untreated or treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or AO / 100 μM bafilomycin A ( BfnA1 ) for 6 hours in the presence of biotin . Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins . Proteins in whole cell extracts and pulldowns from each condition were then separated by SDS - PAGE and membranes probed with specific antibodies . Immunoblotting for Tom1 and mycBioID - Tollip were used as positive controls . Cells cultured in media lacking biotin were used as a negative control to assess background levels . Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions . ( B ) HeLa cells expressing mycBioID alone and HA - Parkin were used to confirm the specificity of the Tollip - Parkin interaction . ( C ) Hela cells co - expressing GFP - Tollip and HA - Parkin were either left untreated or treated with AO for 2 hours prior to lysis . Cell lysates were subjected to either a normal rabbit IgG IP or HA IP and western blot analysis was performed using antibodies against Parkin and GFP . ( D ) HeLa cells expressing mycBioID - Tollip containing the CUE domain mutation ( CUEmut ) and HA - Parkin were subjected to biotinylation and streptavidin pulldowns , followed by western blot analysis . ( E ) HeLa wild - type ( WT ) , Tom1 KO and ATG5 KO cells expressing mycBioID - Tollip and HA - Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins .

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Figure 3(A) HeLa cells expressing mycBioID-Tollip and HA-Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 hours in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole cell extracts and pulldowns from each condition were then separated by SDS-PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID-Tollip were used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions.(B) HeLa cells expressing mycBioID alone and HA-Parkin were used to confirm the specificity of the Tollip-Parkin interaction.(C) Hela cells co-expressing GFP-Tollip and HA-Parkin were either left untreated or treated with AO for 2 hours prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP and western blot analysis was performed using antibodies against Parkin and GFP.(D) HeLa cells expressing mycBioID-Tollip containing the CUE domain mutation (CUEmut) and HA-Parkin were subjected to biotinylation and streptavidin pulldowns, followed by western blot analysis.(E) HeLa wild-type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID-Tollip and HA-Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins.


Figure 4 ( A ) Cells were reverse transfected with Parkin or Tom1 siRNA oligos for 24 hours before a second transfection was performed for a further 48 hours , then fixed and stained with antibodies specific to TOM20 ( green ) and PDH E2 / E3bp ( red ) . Nuclei are labelled with Hoechst ( blue ) . Zoom insets represent 5x magnification and arrowheads indicate TOM20 - positive MDVs . ( B ) Knockdowns were confirmed for both Parkin and Tom1 by western blotting . ( C , D ) Quantification of MDVs , defined as TOM20 + ve / PDH - ve , ( C ) or PDH + ve / TOM20 - ve ( D ) per cell under steady - state conditions were performed ( n = 32 - 52 cells per condition from 3 independent experiments ) . ( E ) Western blot analysis of lysates harvested from SH - SY5Y wild - type ( empty vector ) and CRISPR generated Parkin KO cells . Immunoblot analysis was performed using antibodies against Parkin and GAPDH . ( F ) Immunofluorescence microscopy was performed on SH - SY5Y wild - type and Parkin KO cells either untreated or treated with AO for 2 hours . Cells were immunostained for TOM20 ( red ) , PDH E2 / E3bp ( blue ) , and LAMP1 ( green ) . Zoom insets represent 5x magnification . Circles indicate TOM20 MDVs positive for LAMP1 and arrowheads indicate TOM20 MDVs negative for LAMP1 . Scale bar represents 10 µm . ( G ) Quantitation of TOM20 - positive MDV colocalisation with LAMP1 were performed across all treatment groups and represented as the % of TOM20 - positive MDVs colocalising with LAMP1 . Results represent 3 independent experiments ( 28 - 53 cells / condition ) and statistical significance was determined using a Two - way ANOVA ( Sidak ) . ( H ) HeLa cells transiently transfected with HA - Parkin were subjected to mitochondrial stress by treatment with 25 μM antimycin A ( AA ) for 2 hours , then fixed and stained with antibodies specific to HA , PDH and TOM20 . TOM20 + ve / PDH - ve MDVs were quantified in cells that were either expressing or not expressing HA - Parkin ( n = 12 - 14 cells per condition from 3 independent experiments ) . ( I ) Hela cells stably coexpressing BioID - Tollip and either HA - Parkin WT or Δ1 - 76 ( ΔUBL ) were incubated with and without biotin in the presence of AO for 6 hours prior to being subjected to cell lysis and streptavidin pulldowns . Western blot analysis was performed on lysate inputs and streptavidin pulldowns ( Streptavidin PD ) using antibodies against the indicated proteins or epitope tags .

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Figure 4(A) Cells were reverse transfected with Parkin or Tom1 siRNA oligos for 24 hours before a second transfection was performed for a further 48 hours, then fixed and stained with antibodies specific to TOM20 (green) and PDH E2/E3bp (red). Nuclei are labelled with Hoechst (blue). Zoom insets represent 5x magnification and arrowheads indicate TOM20-positive MDVs.(B) Knockdowns were confirmed for both Parkin and Tom1 by western blotting.(C, D) Quantification of MDVs, defined as TOM20+ve/PDH-ve, (C) or PDH+ve/TOM20-ve (D) per cell under steady-state conditions were performed (n = 32-52 cells per condition from 3 independent experiments).(E) Western blot analysis of lysates harvested from SH-SY5Y wild-type (empty vector) and CRISPR generated Parkin KO cells. Immunoblot analysis was performed using antibodies against Parkin and GAPDH.(F) Immunofluorescence microscopy was performed on SH-SY5Y wild-type and Parkin KO cells either untreated or treated with AO for 2 hours. Cells were immunostained for TOM20 (red), PDH E2/E3bp (blue), and LAMP1 (green). Zoom insets represent 5x magnification. Circles indicate TOM20 MDVs positive for LAMP1 and arrowheads indicate TOM20 MDVs negative for LAMP1. Scale bar represents 10 µm.(G) Quantitation of TOM20-positive MDV colocalisation with LAMP1 were performed across all treatment groups and represented as the % of TOM20-positive MDVs colocalising with LAMP1. Results represent 3 independent experiments (28-53 cells/condition) and statistical significance was determined using a Two-way ANOVA (Sidak).(H) HeLa cells transiently transfected with HA-Parkin were subjected to mitochondrial stress by treatment with 25 μM antimycin A (AA) for 2 hours, then fixed and stained with antibodies specific to HA, PDH and TOM20. TOM20+ve/PDH-ve MDVs were quantified in cells that were either expressing or not expressing HA-Parkin (n = 12-14 cells per condition from 3 independent experiments).(I) Hela cells stably coexpressing BioID-Tollip and either HA-Parkin WT or Δ1-76 (ΔUBL) were incubated with and without biotin in the presence of AO for 6 hours prior to being subjected to cell lysis and streptavidin pulldowns. Western blot analysis was performed on lysate inputs and streptavidin pulldowns (Streptavidin PD) using antibodies against the indicated proteins or epitope tags.


Figure 5 ( A - B ) siRNA knockdown of Tollip or Parkin in SH - SY5Y cells was performed over a 72 hour period , then 18 hours prior to treatment cells were transfected with GFP - 2xFYVE to label endocytic membranes . Cells were treated with 25 μM antimycin A ( AA ) for 2 hours then fixed and immunostained with antibodies specific to PDH E2 / E3bp ( red ) and TOM20 ( blue ) . Arrowheads denote TOM20 + ve / PDH - ve MDVs that colocalise with GFP - 2xFYVE and arrows denote TOM20 + ve / PDH - ve MDVs that do not . ( C ) Quantification of TOM20 + ve / PDH - ve MDVs that are positive for GFP - 2xFYVE are shown as a % of TOM20 MDVs positive for GFP , with each data point indicating 1 cell . MDV colocalisation were quantified by eye from images captured ( n = 20 - 22 cells per condition from 3 independent experiments ) . ( D ) SH - SY5Y cells were left untreated or treated with 25 μM AA for 2 hours prior to fixation and immunostaining for EEA1 ( green ) , PDH E2 / E3bp ( red ) , and TOM20 ( blue ) . Arrowheads indicate TOM20 + ve / PDH - ve MDVs positive for the early endosome marker EEA1 . ( E ) siRNA knockdown of Parkin was performed in SH - SY5Y cells followed by 25 μM AA for 2 hours prior to fixation and immunostaining with antibodies specific to TOM20 ( green ) , Tollip ( red ) and Cytochrome c ( blue ) . We observed Tollip colocalisation with TOM20 + ve / PDH - ve MDVs ( denoted by arrowheads ) , which was still maintained following Parkin siRNA knockdown . ( F ) SH - SY5Y cells were transfected with GFP - Tollip and treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or AO / 100 μM bafilomycin A ( BfnA1 ) for 2 hours prior to fixation and immunostaining with antibodies specific to GFP ( green ) , Rab7a ( red ) and PDH E2 / E3bp ( blue ) . ( G ) Quantification of GFP - Tollip / Rab7a colocalisation using Pearson ' s correlation .

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Figure 5(A-B) siRNA knockdown of Tollip or Parkin in SH-SY5Y cells was performed over a 72 hour period, then 18 hours prior to treatment cells were transfected with GFP-2xFYVE to label endocytic membranes. Cells were treated with 25 μM antimycin A (AA) for 2 hours then fixed and immunostained with antibodies specific to PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs that colocalise with GFP-2xFYVE and arrows denote TOM20+ve/PDH-ve MDVs that do not.(C) Quantification of TOM20+ve/PDH-ve MDVs that are positive for GFP-2xFYVE are shown as a % of TOM20 MDVs positive for GFP, with each data point indicating 1 cell. MDV colocalisation were quantified by eye from images captured (n = 20-22 cells per condition from 3 independent experiments).(D) SH-SY5Y cells were left untreated or treated with 25 μM AA for 2 hours prior to fixation and immunostaining for EEA1 (green), PDH E2/E3bp (red), and TOM20 (blue). Arrowheads indicate TOM20+ve/PDH-ve MDVs positive for the early endosome marker EEA1.(E) siRNA knockdown of Parkin was performed in SH-SY5Y cells followed by 25 μM AA for 2 hours prior to fixation and immunostaining with antibodies specific to TOM20 (green), Tollip (red) and Cytochrome c (blue). We observed Tollip colocalisation with TOM20+ve/PDH-ve MDVs (denoted by arrowheads), which was still maintained following Parkin siRNA knockdown.(F) SH-SY5Y cells were transfected with GFP-Tollip and treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 2 hours prior to fixation and immunostaining with antibodies specific to GFP (green), Rab7a (red) and PDH E2/E3bp (blue). (G) Quantification of GFP-Tollip/Rab7a colocalisation using Pearson's correlation.


Figure 6 ( A ) Quantitation of Tollip vesicular clusters in SH - SY5Ys treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) for 2 hours . The percentage of cells with vesicular clusters were quantified from 7 - 23 cells for each condition from 4 independent experiments . ( B ) Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 hours . The percentage of cells with vesicular clusters were quantified from an average of 50 cells for each condition from 5 independent experiments . ( C ) Endogenous Tollip and LAMP1 colocalise in SH - SY5Y cells treated with AO for 2 hours . Antibodies specific to LAMP1 ( green ) and Tollip ( red ) were used . ( D - E ) SH - SY5Y cells were transfected with either wild - type GFP - Tollip ( WT ) , GFP - Tollip lacking its N - terminus ( ∆ Nterm ) , or GFP - Tollip containing a CUE domain mutation ( CUEmut ) then treated with AO for 2 hours . Cells were fixed and immunostained with antibodies specific to GFP ( green ) and LAMP1 ( red ) . ( F ) The extent of colocalisation between GFP - Tollip and LAMP1 from untreated and AO treated SH - SY5Y cells were quantitated from immunofluorescence images and represented as a Pearson ' s correlation ( 2 - 3 cells per experiment , from 2 independent experiments ) . ( G - I ) The extent of colocalisation between GFP - Tollip and LAMP1 in Tom1 KO ( G ) , ATG5 KO ( H ) , and VPS35 KO ( I ) compared to wild - type ( WT ) HeLa cells was quantified from immunofluorescence images immunostained for GFP and LAMP1 and represented as a Pearson ' s correlation ( 2 - 3 cells per experiment , from 3 independent experiments ) . Western blot analysis of lysates harvested from wild - type ( WT ) alongside either Tom1 KO ( G ) or VPS35 KO ( I ) Hela cells was performed using antibodies specific to the indicated proteins .

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Figure 6(A) Quantitation of Tollip vesicular clusters in SH-SY5Ys treated with 5 μM antimycin A/10 μM oligomycin B (AO) for 2 hours. The percentage of cells with vesicular clusters were quantified from 7-23 cells for each condition from 4 independent experiments.(B) Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 hours. The percentage of cells with vesicular clusters were quantified from an average of 50 cells for each condition from 5 independent experiments.(C) Endogenous Tollip and LAMP1 colocalise in SH-SY5Y cells treated with AO for 2 hours. Antibodies specific to LAMP1 (green) and Tollip (red) were used.(D-E) SH-SY5Y cells were transfected with either wild-type GFP-Tollip (WT), GFP-Tollip lacking its N-terminus (∆Nterm), or GFP-Tollip containing a CUE domain mutation (CUEmut) then treated with AO for 2 hours. Cells were fixed and immunostained with antibodies specific to GFP (green) and LAMP1 (red).(F) The extent of colocalisation between GFP-Tollip and LAMP1 from untreated and AO treated SH-SY5Y cells were quantitated from immunofluorescence images and represented as a Pearson's correlation (2-3 cells per experiment, from 2 independent experiments). (G-I) The extent of colocalisation between GFP-Tollip and LAMP1 in Tom1 KO (G), ATG5 KO (H), and VPS35 KO (I) compared to wild-type (WT) HeLa cells was quantified from immunofluorescence images immunostained for GFP and LAMP1 and represented as a Pearson's correlation (2-3 cells per experiment, from 3 independent experiments). Western blot analysis of lysates harvested from wild-type (WT) alongside either Tom1 KO (G) or VPS35 KO (I) Hela cells was performed using antibodies specific to the indicated proteins.


Figure 7 ( A ) A subset of TOM20 + ve / PDH - ve MDVs are trafficked to the lysosome . siRNA knockdown of Tollip was conducted in SH - SY5Y cells over a 72 hours period prior to treatment with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or 25 μM antimycin A ( AA ) for 2 hours . Cells were then fixed and stained with antibodies specific to LAMP1 ( green ) , PDH E2 / E3bp ( red ) and TOM20 ( blue ) . Arrows denote TOM20 + ve / PDH - ve MDVs colocalising with LAMP1 , whereas arrowheads denote MDVs that do not colocalise with LAMP1 . ( B ) From immunofluorescence images , the total number of TOM20 + ve / PDH - ve MDVs were counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20 positive MDVs colocalising with LAMP1 per cell ( n = 25 - 32 cells per condition from 3 independent experiments ) . ( C ) siRNA knockdown was performed in SH - SY5Y cells , then 18 hours prior to fixation cells were transfected with GFP - Tollip wild - type ( WT ) , ∆ Nterm , CUEmut , or R78A . Cells were fixed and immunostained for PDH E2 / E3bp ( red ) and TOM20 ( blue ) . Arrowheads denote TOM20 + ve / PDH - ve MDVs . ( D ) Quantification of TOM20 MDVs per cell was performed by eye ( n = 11 - 14 cells per mutant from 2 independent experiments ) . Cells expressing a GFP - Tollip construct were identified using the GFP channel . Surrounding cells not expressing GFP were counted as the Tollip siRNA only population .

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Figure 7(A) A subset of TOM20+ve/PDH-ve MDVs are trafficked to the lysosome. siRNA knockdown of Tollip was conducted in SH-SY5Y cells over a 72 hours period prior to treatment with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM antimycin A (AA) for 2 hours. Cells were then fixed and stained with antibodies specific to LAMP1 (green), PDH E2/E3bp (red) and TOM20 (blue). Arrows denote TOM20+ve/PDH-ve MDVs colocalising with LAMP1, whereas arrowheads denote MDVs that do not colocalise with LAMP1. (B) From immunofluorescence images, the total number of TOM20+ve/PDH-ve MDVs were counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20 positive MDVs colocalising with LAMP1 per cell (n = 25-32 cells per condition from 3 independent experiments).(C) siRNA knockdown was performed in SH-SY5Y cells, then 18 hours prior to fixation cells were transfected with GFP-Tollip wild-type (WT), ∆Nterm, CUEmut, or R78A. Cells were fixed and immunostained for PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs. (D) Quantification of TOM20 MDVs per cell was performed by eye (n = 11-14 cells per mutant from 2 independent experiments). Cells expressing a GFP-Tollip construct were identified using the GFP channel. Surrounding cells not expressing GFP were counted as the Tollip siRNA only population.


Figure 1Heatmap showing the normalized expression of marker genes for the different clusters ( Methods ) . Expression is normalized by the maximal expression among all cell types .

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Figure 1Heatmap showing the normalized expression of marker genes for the different clusters (Methods). Expression is normalized by the maximal expression among all cell types.


Figure 2Top - left - tSNE plot of the Seurat clusters , boxes demarcate compared clusters . Dashed boxes and labels indicate the cell clusters that are compared in panels A - C . ( A ) Volcano plot of differential gene expression ( DGE ) between liver vascular endothelial cells in the tumor and non - tumor samples . ( B ) Volcano plot of DGE between mononuclear phagocytes in the malignant and non - malignant samples . ( C ) Volcano plot of DGE between T cells in the tumor and non - tumor samples . ( D ) DGE analysis between tumor mononuclear phagocytes classified by cancer type ( cholangiocarcinoma in dark purple and metastasis in light purple ) . Wilcoxon rank - sum tests were used to generate p - values , Benjamini - Hochberg multiple hypotheses correction was used to compute q - values . Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two clusters .

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Figure 2Top-left - tSNE plot of the Seurat clusters, boxes demarcate compared clusters. Dashed boxes and labels indicate the cell clusters that are compared in panels A-C. (A) Volcano plot of differential gene expression (DGE) between liver vascular endothelial cells in the tumor and non-tumor samples. (B) Volcano plot of DGE between mononuclear phagocytes in the malignant and non-malignant samples. (C) Volcano plot of DGE between T cells in the tumor and non-tumor samples. (D) DGE analysis between tumor mononuclear phagocytes classified by cancer type (cholangiocarcinoma in dark purple and metastasis in light purple). Wilcoxon rank-sum tests were used to generate p-values, Benjamini-Hochberg multiple hypotheses correction was used to compute q-values. Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two clusters.


Figure 3Key marker genes for the four mesenchymal clusters ( RBP1 , COL1A1 , RGS5 , and MYH11 ) . Light gray dots denote cells originating from non - tumor samples . Dark gray dots denote cells originating from the tumor samples . Left - Representative smFISH image of patient p1 stained for RGS5 and COL1A1 showing distinct spatial localization of CAFs and Pericytes . Scale bar 10µm . Dashed lines mark the shortest distance of cells ( 2a ) and ( 2b ) from the cell ( 1 ) . Middle - zoom - in of ( 1 ) from left panel , showing a blood vessel like structure formed by endothelial cells marked by PDGFB ( magenta ) wrapped by pericytes marked by RGS5 ( green ) . Dashed lines are two consecutive cell layers of endothelial cells and pericytes . Scale bar 2 . 5µm . Right - zoom - in of ( 2a and b ) from the left panel , showing two distant CAFs expressing high COL1A1 signal but not RGS5 . DAPI used for nuclei staining . Scale bar 2 . 5µm . Violin plot of the distance from blood vessels of low / high RGS5 expressing cells ( n = 358 and n = 360 respectively ) and low / high COL1A1 expressing cells ( n = 359 and n = 359 respectively ) . ' p ' is the p - value determined by Wilcoxon rank - sum test . Empty circles are the medians over all repeats .

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Figure 3Key marker genes for the four mesenchymal clusters (RBP1, COL1A1, RGS5, and MYH11). Light gray dots denote cells originating from non-tumor samples. Dark gray dots denote cells originating from the tumor samples. Left - Representative smFISH image of patient p1 stained for RGS5 and COL1A1 showing distinct spatial localization of CAFs and Pericytes. Scale bar 10µm. Dashed lines mark the shortest distance of cells (2a) and (2b) from the cell (1). Middle - zoom-in of (1) from left panel, showing a blood vessel like structure formed by endothelial cells marked by PDGFB (magenta) wrapped by pericytes marked by RGS5 (green). Dashed lines are two consecutive cell layers of endothelial cells and pericytes. Scale bar 2.5µm. Right - zoom-in of (2a and b) from the left panel, showing two distant CAFs expressing high COL1A1 signal but not RGS5. DAPI used for nuclei staining. Scale bar 2.5µm.Violin plot of the distance from blood vessels of low/high RGS5 expressing cells (n=358 and n= 360 respectively) and low/high COL1A1 expressing cells (n=359 and n= 359 respectively). 'p' is the p-value determined by Wilcoxon rank-sum test. Empty circles are the medians over all repeats.


Figure 4Dot - plot of selected genes highlight shared interaction motifs between different clusters colored by max normalized expression for each genes across all clusters in ( A ) . For each gene , dot size represents the fraction of positive cells for each cluster .

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Figure 4Dot-plot of selected genes highlight shared interaction motifs between different clusters colored by max normalized expression for each genes across all clusters in (A). For each gene, dot size represents the fraction of positive cells for each cluster.


Figure 5H & E staining of malignant and non - malignant sites from patient p4 , separated by the white dashed line . Scale bar is 100µm . Non - malignant human liver lobule from patient p4 used for LCM stained with HistoGene Staining Solution ( Methods ) . Top - sequential zones from the central vein ( cv ) to the portal node ( pn ) before microdissection . LCM captured zones marked by the black dashed line . White dashed line marks the boundaries of NPC ( non - parenchymal cells ) located in the pn and not included in the analysis . Bottom - Tissue after microdissection . Scale bar is 100µm . Zonation of human hepatocytes . Left - Max normalized expression over the reconstructed six lobule layers ( L1 to L6 ) , sorted by center of mass from the central vein ( cv ) to the portal node ( pn ) . Right - selected pericentrally ( top ) , and periportally ( bottom ) zonated genes . Blue patches are standard errors of the mean . Zonation profiles of selected genes from human ( blue ) and mouse ( green ) . Each profile is normalized by the maximal expression across zones . Patches are standard errors of the mean .

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

Figure 5H&E staining of malignant and non-malignant sites from patient p4, separated by the white dashed line. Scale bar is 100µm. Non-malignant human liver lobule from patient p4 used for LCM stained with HistoGene Staining Solution (Methods). Top - sequential zones from the central vein (cv) to the portal node (pn) before microdissection. LCM captured zones marked by the black dashed line. White dashed line marks the boundaries of NPC (non-parenchymal cells) located in the pn and not included in the analysis. Bottom - Tissue after microdissection. Scale bar is 100µm.Zonation of human hepatocytes. Left - Max normalized expression over the reconstructed six lobule layers (L1 to L6), sorted by center of mass from the central vein (cv) to the portal node (pn). Right - selected pericentrally (top), and periportally (bottom) zonated genes. Blue patches are standard errors of the mean.Zonation profiles of selected genes from human (blue) and mouse (green). Each profile is normalized by the maximal expression across zones. Patches are standard errors of the mean.


Figure 6tissue sections from patients p1 and p3 stained with HistoGene Staining Solution . Laser captured zones are marked with dashed black line including : tumor core ( tc ) , the border between the tumor and the fibrotic regions ( ftb ) , tumor islets within the fibrotic regions ( ti ) and fibrotic zones ( fz ) in panel ( A ) , and tumor border ( tb ) in panel ( B ) . Small images in ( A ) are magnifications of the corresponding zones in the large image for the left . White dashed line in ( B ) marking the non - malignant liver ( nml ) border . Scale bars in all panels are 100µm .

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Figure 6tissue sections from patients p1 and p3 stained with HistoGene Staining Solution. Laser captured zones are marked with dashed black line including: tumor core (tc), the border between the tumor and the fibrotic regions (ftb), tumor islets within the fibrotic regions (ti) and fibrotic zones (fz) in panel (A), and tumor border (tb) in panel (B). Small images in (A) are magnifications of the corresponding zones in the large image for the left. White dashed line in (B) marking the non-malignant liver (nml) border. Scale bars in all panels are 100µm.


Figure 1A ) M . florum doubling time at different incubation temperatures measured by colorimetric assays . The bars represent the mean and standard deviation values obtained from three technical replicates . The asterisk indicates the absence of significant growth , preventing the calculation of a doubling time . D ) Representative images of SYTO 9 and propidium iodide ( PI ) double stained M . florum cells , harvested from an exponential or death - phase culture , observed by widefield fluorescence microscopy . The brightness of each channel was adjusted equally between conditions . Scale bar : 5 µm .

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Figure 1A) M. florum doubling time at different incubation temperatures measured by colorimetric assays. The bars represent the mean and standard deviation values obtained from three technical replicates. The asterisk indicates the absence of significant growth, preventing the calculation of a doubling time.D) Representative images of SYTO 9 and propidium iodide (PI) double stained M. florum cells, harvested from an exponential or death-phase culture, observed by widefield fluorescence microscopy. The brightness of each channel was adjusted equally between conditions. Scale bar: 5 µm.


Figure 2A ) Representative image of M . florum cells observed by transmission electronic microscopy ( TEM ) . Scale : 100 nm . D ) Boxplots showing the median and interquartile range of the average cell diameter calculated from 389 and 169 individual cells analyzed by TEM and STED , respectively . Whiskers indicate the 10 - 90 percentile range .

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Figure 2A) Representative image of M. florum cells observed by transmission electronic microscopy (TEM). Scale: 100 nm.D) Boxplots showing the median and interquartile range of the average cell diameter calculated from 389 and 169 individual cells analyzed by TEM and STED, respectively. Whiskers indicate the 10-90 percentile range.


Figure 3A ) Circular representation of the M . florum L1 chromosome enhanced with 5 ' - RACE data generated in this study . Outer to inner circle : genomic coordinates ( kbp ) ; genes encoded on the positive ( blue for coding sequences , turquoise for RNAs ) and negative ( red for coding sequences , fuchsia for RNAs ) DNA strands ; raw 5 ' - RACE signal ( 0 - 1 , 000 read starts scale ) observed at each genomic position for the positive ( black ) and negative ( gray ) DNA strands ; putative transcription start sites ( TSSs ) identified on the positive ( green ) and negative ( orange ) DNA strands from significant 5 ' - RACE peaks .

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Figure 3A) Circular representation of the M. florum L1 chromosome enhanced with 5'-RACE data generated in this study. Outer to inner circle: genomic coordinates (kbp); genes encoded on the positive (blue for coding sequences, turquoise for RNAs) and negative (red for coding sequences, fuchsia for RNAs) DNA strands; raw 5'-RACE signal (0-1,000 read starts scale) observed at each genomic position for the positive (black) and negative (gray) DNA strands; putative transcription start sites (TSSs) identified on the positive (green) and negative (orange) DNA strands from significant 5'-RACE peaks.


Figure 5A ) Transcription levels of all M . florum coding sequences ( CDS ) quantified by RNA - seq . Transcription levels were calculated according to the number of fragments per kilobase per million of mapped reads ( FPKM ) observed over six replicates . The corresponding numbers of mRNA copies per cell , estimated from the measured M . florum RNA mass , are also indicated . CDS were sorted from least to most transcribed . The transcription level of selected genes of importance are presented . LDH , L - lactate dehydrogenase ( peg . 600 / mfl596 ) ; G3PDH , glyceraldehyde - 3 - phosphate dehydrogenase ( peg . 583 / mfl578 ) ; PGK , phosphoglycerate kinase ( peg . 582 / mfl577 ) ; L24p and L7 / L12 , large subunit ribosomal proteins L24p ( peg . 133 / mfl134 ) and L7 / L12 ( peg . 605 / mfl601 ) ; S7p , small subunit ribosomal protein S7p ( peg . 626 / mfl623 ) ; RNAPβ , RNAPβ ' , and RNAPα , RNA polymerase subunits β , β ' , and α ( peg . 601 / mfl597 , peg . 602 / mfl598 , and peg . 149 / mfl150 ) ; HPr , phosphotransferase system phosphocarrier protein HPr ( peg . 570 / mfl565 ) ; σ70 , RNA polymerase sigma factor RpoD ( peg . 269 / mfl270 ) . C ) Expression levels of all M . florum reference proteins quantified by two - dimensional liquid chromatography - tandem mass spectrometry ( 2D LC - MS / MS ) . Abundance was estimated according to the normalized spectral abundance factor ( NSAF ) calculated for each protein . A NSAF value of 0 was assigned to undetected proteins . The corresponding number of protein molecules per cell ( derived from the biomass data ) are indicated . Proteins were sorted from least to most abundant . The selected genes of importance presented in panel A are also highlighted .

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Figure 5A) Transcription levels of all M. florum coding sequences (CDS) quantified by RNA-seq. Transcription levels were calculated according to the number of fragments per kilobase per million of mapped reads (FPKM) observed over six replicates. The corresponding numbers of mRNA copies per cell, estimated from the measured M. florum RNA mass, are also indicated. CDS were sorted from least to most transcribed. The transcription level of selected genes of importance are presented. LDH, L-lactate dehydrogenase (peg.600/mfl596); G3PDH, glyceraldehyde-3-phosphate dehydrogenase (peg.583/mfl578); PGK, phosphoglycerate kinase (peg.582/mfl577); L24p and L7/L12, large subunit ribosomal proteins L24p (peg.133/mfl134) and L7/L12 (peg.605/mfl601); S7p, small subunit ribosomal protein S7p (peg.626/mfl623); RNAPβ, RNAPβ', and RNAPα, RNA polymerase subunits β, β', and α (peg.601/mfl597, peg.602/mfl598, and peg.149/mfl150); HPr, phosphotransferase system phosphocarrier protein HPr (peg.570/mfl565); σ70, RNA polymerase sigma factor RpoD (peg.269/mfl270).C) Expression levels of all M. florum reference proteins quantified by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Abundance was estimated according to the normalized spectral abundance factor (NSAF) calculated for each protein. A NSAF value of 0 was assigned to undetected proteins. The corresponding number of protein molecules per cell (derived from the biomass data) are indicated. Proteins were sorted from least to most abundant. The selected genes of importance presented in panel A are also highlighted.


Figure 2 ( B ) In vitro degradation of UbLRad23 - GFP - 95 by purified S . cerevisiae proteasome . The graph plots the amount of substrate over time , estimated by fluorescence intensity monitored by plate - reader ( green ) or electronic autoradiography of SDS - PAGE gel bands ( red ) , normalized to the initial substrate amount as described in Methods . ( C ) Michaelis - Menten analysis of UbLRad23 - GFP - 95 degradation by different concentrations of purified S . cerevisiae proteasome ( green , 10 nM ; red , 40 nM ) . ( D ) UbLRad23 inhibits UbLRad23 - GFP - 95 degradation . The initial degradation rates of UbLRad23 - GFP - 95 at different concentrations of purified UbLRad23 domain are plotted and fitted to the equation describing competitive inhibition . ( E ) The UbLDsk2 domain is a competitive inhibitor of UbLRad23 - GFP - 95 degradation . Lineweaver - Burk plot of UbLRad23GFP95 degradation with different concentrations of purified UbLDsk2 domain . ( C - E ) Proteasomal degradation of UbLRad23 - GFP - 95 monitored by fluorescence intensity in a Tecan plate reader as described in Methods .

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Figure 2(B) In vitro degradation of UbLRad23-GFP-95 by purified S. cerevisiae proteasome. The graph plots the amount of substrate over time, estimated by fluorescence intensity monitored by plate-reader (green) or electronic autoradiography of SDS-PAGE gel bands (red), normalized to the initial substrate amount as described in Methods. (C) Michaelis-Menten analysis of UbLRad23-GFP-95 degradation by different concentrations of purified S. cerevisiae proteasome (green, 10 nM; red, 40 nM). (D) UbLRad23 inhibits UbLRad23-GFP-95 degradation. The initial degradation rates of UbLRad23-GFP-95 at different concentrations of purified UbLRad23 domain are plotted and fitted to the equation describing competitive inhibition. (E) The UbLDsk2 domain is a competitive inhibitor of UbLRad23-GFP-95 degradation. Lineweaver-Burk plot of UbLRad23GFP95 degradation with different concentrations of purified UbLDsk2 domain. (C-E) Proteasomal degradation of UbLRad23-GFP-95 monitored by fluorescence intensity in a Tecan plate reader as described in Methods.


Figure 3 ( A , B ) In vitro degradation of model proteins by purified S . cerevisiae proteasome . The model proteins ( UbL - DHFR - 95 ) consisted of an N - terminal UbL domain , followed by an E . coli DHFR domain and a 95 aminoacid tail derived from S . cerevisiae cytochrome b2 . UbL - DHFR - 95 proteins were degraded by proteasome ( red solid circle ) and can be stabilized by removing the UbL domain ( no UbL , black triangles ) , by removing the 95 - amino - acid tail ( no tail , blue diamonds ) or by proteasome inhibitor ( MG132 , green squares ) . ( A ) Degradation of model proteins containing the UbL domain of S . cerevisiae Rad23 . ( A , B ) In vitro degradation of model proteins by purified S . cerevisiae proteasome . The model proteins ( UbL - DHFR - 95 ) consisted of an N - terminal UbL domain , followed by an E . coli DHFR domain and a 95 aminoacid tail derived from S . cerevisiae cytochrome b2 . UbL - DHFR - 95 proteins were degraded by proteasome ( red solid circle ) and can be stabilized by removing the UbL domain ( no UbL , black triangles ) , by removing the 95 - amino - acid tail ( no tail , blue diamonds ) or by proteasome inhibitor ( MG132 , green squares ) . ( A ) Degradation of model proteins containing the UbL domain of S . cerevisiae Rad23 . ( B ) Degradation of model proteins containing other UbL domains or ubiquitin . The graphs plot the amount of substrate estimated by electronic autoradiography in SDS - PAGE gel bands over time as normalized to the initial substrate amount as described in Methods . Data points represent mean values determined from at least three repeat experiments ; error bars indicate s . e . m .

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Figure 3(A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23.(A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23. (B) Degradation of model proteins containing other UbL domains or ubiquitin. The graphs plot the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.


Figure 4 ( B ) UbL domains mediated degradation of His3 fusion proteins with a Su9 tail , and affected yeast growth under selective condition ( + 3 - AT , - his ) . Replacing UbL domains with DHFR domains rescued the growth defect . Cells expressing His3 fusion proteins in late log phase were serially diluted and stamped on selective plates . Plates were incubated at 30 ˚ C for 3 daysfor imaging .

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

Figure 4(B) UbL domains mediated degradation of His3 fusion proteins with a Su9 tail, and affected yeast growth under selective condition (+3-AT, -his). Replacing UbL domains with DHFR domains rescued the growth defect. Cells expressing His3 fusion proteins in late log phase were serially diluted and stamped on selective plates. Plates were incubated at 30 ˚C for 3 daysfor imaging.


Figure 5 ( B ) Fluorescence profiles of cells expressing different substrates monitored by flow cytometry . UbLRad23 targeted YFP - Su9 protein for degradation . Removing the Su9 tail or replacing UbLRad23 with a DHFR domain stabilizes YFP protein in cells . ( C ) UbL domains targeted YFP substrates to degradation in the presence of an initiation region . The Y - axis plots abundance of the UbL - YFP - tail proteins normalized by a translation control , which is the same UbL - YFP protein without the tail . The ratio is equal to one for domains that are not recognized by the proteasome , and smaller than one if the UbL is recognized by the proteasome and the UbL - YFP - tail protein is degraded . YFP fluorescence is also corrected for plasmid copy number using RFP fluorescence . The median of YFP / RFP ratio for each construct was calculated from 10 , 000 cells collected in one flow cytometry run and reflected the abundance of YFP substrates in yeast cells as described in Methods . Data points represent mean values determined from at least three repeat experiments ; error bars indicate s . e . m .

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 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

Figure 5(B) Fluorescence profiles of cells expressing different substrates monitored by flow cytometry. UbLRad23 targeted YFP-Su9 protein for degradation. Removing the Su9 tail or replacing UbLRad23 with a DHFR domain stabilizes YFP protein in cells.(C) UbL domains targeted YFP substrates to degradation in the presence of an initiation region. The Y-axis plots abundance of the UbL-YFP-tail proteins normalized by a translation control, which is the same UbL-YFP protein without the tail. The ratio is equal to one for domains that are not recognized by the proteasome, and smaller than one if the UbL is recognized by the proteasome and the UbL-YFP-tail protein is degraded. YFP fluorescence is also corrected for plasmid copy number using RFP fluorescence. The median of YFP/RFP ratio for each construct was calculated from 10,000 cells collected in one flow cytometry run and reflected the abundance of YFP substrates in yeast cells as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.


Figure 6 ( B ) In vitro degradation of Mdy2 proteins by purified S . cerevisiae proteasome . Mdy2 ( red solid circles ) , Mdy2ΔN - 95 ( blue solid circles ) and Mdy2 - 95 ( green solid circles ) were degraded . Removing the N - terminal domain of Mdy2 ( Mdy2ΔN , black solid circles ) or adding its binding partner Get4 ( red open squares , dash line ) or the proteasome - binding competitor UbLMdy2 ( red open diamonds , dotted line ) stabilized Mdy2Get4 . When Mdy2 binding buried the N - terminal domain , addition of a 95 amino - acid tail derived from S . cerevisiae cytochrome b2 at the Cterminus restored degradation of Mdy2 ( green open squares , dotted line ) . The graph plots the amount of substrate estimated by electronic autoradiography in SDS - PAGE gel bands over time as normalized to the initial substrate amount as described in Methods . Data points represent mean values determined from at least three repeat experiments ; error bars indicate s . e . m . ( C ) Degradation of Mdy2 in vivo . The steady - state accumulation of overexpressed C - terminally HA - tagged full - length Mdy2 and a mutant in which the N - terminal domain was deleted ( Mdy2ΔN ) in S . cerevisiae , in the absence of Get4 , the presence of endogenously expressed Get4 or overexpressed Get4 . Overexpressed Get4 was detected through an N - terminal Flag - tag . ( D ) Steady - state accumulation of endogenous Mdy2 in S . cerevisiae , with deleted ( get4 ∆ ) or endogenous ( wt ) Get4 in the presence or absence of the proteasome inhibitor bortezomib . Protein levels were determined by western blotting for the HA - tag , Flag - tag , Mdy2 or Scs2 of SDS - PAGE gels of S . cerevisiae protein extracts . Protein loading levels in each lane were estimated in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2 .

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

Figure 6(B) In vitro degradation of Mdy2 proteins by purified S. cerevisiae proteasome. Mdy2 (red solid circles), Mdy2ΔN-95 (blue solid circles) and Mdy2-95 (green solid circles) were degraded. Removing the N-terminal domain of Mdy2 (Mdy2ΔN, black solid circles) or adding its binding partner Get4 (red open squares, dash line) or the proteasome-binding competitor UbLMdy2 (red open diamonds, dotted line) stabilized Mdy2Get4. When Mdy2 binding buried the N-terminal domain, addition of a 95 amino-acid tail derived from S. cerevisiae cytochrome b2 at the Cterminus restored degradation of Mdy2 (green open squares, dotted line). The graph plots the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.(C) Degradation of Mdy2 in vivo. The steady-state accumulation of overexpressed C-terminally HA-tagged full-length Mdy2 and a mutant in which the N-terminal domain was deleted (Mdy2ΔN) in S. cerevisiae, in the absence of Get4, the presence of endogenously expressed Get4 or overexpressed Get4. Overexpressed Get4 was detected through an N-terminal Flag-tag.(D) Steady-state accumulation of endogenous Mdy2 in S. cerevisiae, with deleted (get4∆) or endogenous (wt) Get4 in the presence or absence of the proteasome inhibitor bortezomib. Protein levels were determined by western blotting for the HA-tag, Flag-tag, Mdy2 or Scs2 of SDS-PAGE gels of S. cerevisiae protein extracts. Protein loading levels in each lane were estimated in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.


Figure 7 ( B ) In vitro degradation of Ubp6 proteins by purified S . cerevisiae proteasome . Full - length Ubp6 ( Ubp6 , black open circles ) and a UbL deletion mutant ( Ubp6ΔN , green open circles ) escaped proteasome degradation . Attachment of a Cterminal 95 amino - acid tail derived from S . cerevisiae cytochrome b2 allowed the proteasome to degrade the Ubp6 proteins ( Ubp6 - 95 , black solid circles and Ubp6ΔN - 95 , green solid circles ) . The graph plots the amount of substrate estimated by electronic autoradiography in SDS - PAGE gel bands over time as normalized to the initial substrate amount as described in Methods . Data points represent mean values determined from at least three repeat experiments ; error bars indicate s . e . m . C ) Degradation of Ubp6 proteinsin vivo . The steady - state accumulation of N - terminally HA - tagged Ubp6 and mutants in S . cerevisiae . Ubp6 and Ubp6ΔN are stable in yeast and not further stabilized by bortezomib . Attachment of a Su9 tail to the Cterminus lead to the degradation of Ubp6 and Ubp6ΔNproteins and the proteins were stabilized by bortezomib treatment . ( D ) The steady - state accumulation of endogenous Ubp6 and Ubp6 derivatives with short ( 19 amino acid ) or long ( 54 amino acid ) tails at their C - termini . The 19 amino - acid tail destabilized Ubp6 and the 54 - amino - acid tail lead to complete depletion of Ubp6 . Protein levels were determined by western blotting for the HA - tag , Ubp6 or Scs2 in SDS - PAGE gels of S . cerevisiae protein extracts . The proteasome was inhibited by 100 µM bortezomib as indicated . Protein loading levels in each lane were normalized in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2 .

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

Figure 7(B) In vitro degradation of Ubp6 proteins by purified S. cerevisiae proteasome. Full-length Ubp6 (Ubp6, black open circles) and a UbL deletion mutant (Ubp6ΔN, green open circles) escaped proteasome degradation. Attachment of a Cterminal 95 amino-acid tail derived from S. cerevisiae cytochrome b2 allowed the proteasome to degrade the Ubp6 proteins (Ubp6-95, black solid circles and Ubp6ΔN-95, green solid circles). The graph plots the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.C) Degradation of Ubp6 proteinsin vivo. The steady-state accumulation of N-terminally HA-tagged Ubp6 and mutants in S. cerevisiae. Ubp6 and Ubp6ΔN are stable in yeast and not further stabilized by bortezomib. Attachment of a Su9 tail to the Cterminus lead to the degradation of Ubp6 and Ubp6ΔNproteins and the proteins were stabilized by bortezomib treatment.(D) The steady-state accumulation of endogenous Ubp6 and Ubp6 derivatives with short (19 amino acid) or long (54 amino acid) tails at their C-termini. The 19 amino-acid tail destabilized Ubp6 and the 54-amino-acid tail lead to complete depletion of Ubp6. Protein levels were determined by western blotting for the HA-tag, Ubp6 or Scs2 in SDS-PAGE gels of S. cerevisiae protein extracts. The proteasome was inhibited by 100 µM bortezomib as indicated. Protein loading levels in each lane were normalized in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.


Figure 1 B An Integrative Genomics Viewer ( IGV ) screenshot of the Notch de novo TE insertion site from sample P47 ( clonal neoplasia ) and its head control , sample P48 . Bars represent sequencing reads . Reads supporting the TE insertion are colored according to homology to a specific TE insertion sequence . Multiple colors at a putative insert site frequently indicate homology to different reference copies of the same TE family . Two types of supporting reads can be seen : soft - clipped reads - spanning the insertion site and mapping partially to the reference genome and partially to the TE , and mate pair support reads - flanking the insertion site and mapping to the reference genome but with mates ( not seen ) mapping to the TE . D PCR validation of 4 somatic , neoplasia - specific TE insertions . Primers were designed to target regions flanking the insertion sites . Yellow arrowheads indicate PCR products containing an insertion amplified in the clonal DNA but not in the neighboring gut tissue ( non - clonal ) , head or thorax for the same fly . Short wild - type amplicon was detectable in all samples . Thorax DNA sample was not available for sample P15 .

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

Figure 1 B An Integrative Genomics Viewer (IGV) screenshot of the Notch de novo TE insertion site from sample P47 (clonal neoplasia) and its head control, sample P48. Bars represent sequencing reads. Reads supporting the TE insertion are colored according to homology to a specific TE insertion sequence. Multiple colors at a putative insert site frequently indicate homology to different reference copies of the same TE family. Two types of supporting reads can be seen: soft-clipped reads - spanning the insertion site and mapping partially to the reference genome and partially to the TE, and mate pair support reads - flanking the insertion site and mapping to the reference genome but with mates (not seen) mapping to the TE.  D PCR validation of 4 somatic, neoplasia-specific TE insertions. Primers were designed to target regions flanking the insertion sites. Yellow arrowheads indicate PCR products containing an insertion amplified in the clonal DNA but not in the neighboring gut tissue (non-clonal), head or thorax for the same fly. Short wild-type amplicon was detectable in all samples. Thorax DNA sample was not available for sample P15.


Figure 2 D TSD length distribution for somatic insertions of most frequent TE families . Insertions from both genotypes were pooled .

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

Figure 2 D TSD length distribution for somatic insertions of most frequent TE families. Insertions from both genotypes were pooled.


Figure 3 C The distribution of somatic insertions with estimated cell fraction higher or lower than Notch - inactivating mutations for the most represented LTR - elements ( rover and copia ) and LINE - like I - elements . ( p - values were calculated with Fisher ' s exact test , two - tailed , n = number of insertions )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 C The distribution of somatic insertions with estimated cell fraction higher or lower than Notch-inactivating mutations for the most represented LTR-elements (rover and copia) and LINE-like I-elements. (p-values were calculated with Fisher's exact test, two-tailed, n= number of insertions)


Figure 4 A Heatmaps representing normalized TE expression levels ( in log2 ( TPM ) , transcripts per million ) and mobility ( log2 ( insertion counts ) ) in Pros > 2xGFP midguts . TEs with TPM values below 0 . 05 ( log2 ( TPM ) < - 4 . 3 ) are not depicted . Crossed out cells represent no somatic insertions of that family identified . B Normalized read coverage over the full - length canonical sequence of selected TE families . C Normalized expression levels of siRNA pathway genes . D The size distribution of sense and antisense reads from gut small RNA fractions mapping to all TEs ( upper panel ) or selected TE families mobilizing in the gut . R1 and R2 are two biological replicates . E Scatter plot of normalized transcript ( mRNA ) levels and antisense , 21nt siRNA levels for all TE families . Transposons generating somatic insertions are highlighted in red ( LTR - elements ) or blue ( LINE - like ) , with a symbol size reflecting the somatic insertion counts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heatmaps representing normalized TE expression levels (in log2(TPM), transcripts per million) and mobility (log2(insertion counts)) in Pros>2xGFP midguts. TEs with TPM values below 0.05 (log2(TPM)< -4.3) are not depicted. Crossed out cells represent no somatic insertions of that family identified.  B Normalized read coverage over the full-length canonical sequence of selected TE families.  C Normalized expression levels of siRNA pathway genes.  D The size distribution of sense and antisense reads from gut small RNA fractions mapping to all TEs (upper panel) or selected TE families mobilizing in the gut. R1 and R2 are two biological replicates.  E Scatter plot of normalized transcript (mRNA) levels and antisense, 21nt siRNA levels for all TE families. Transposons generating somatic insertions are highlighted in red (LTR-elements) or blue (LINE-like), with a symbol size reflecting the somatic insertion counts.


Figure 5 B TE families with tissue - specific , singleton TSD - bearing insertions detected in pooled gut or head DNA .

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 B TE families with tissue-specific, singleton TSD-bearing insertions detected in pooled gut or head DNA.


Figure 6 B Somatic TE insertion sites of rover LTR elements were depleted from intergenic and exonic sequences and enriched in introns and 3 ' UTR regions of the fly genome . Insertions of copia LTR elements were also enriched in introns . D Correlations of somatic insertion sites of the three most represented TE families ( rover , copia and I - element ) with modENCODE tracks for adult fly tissues . E Correlations of somatic insertion sites of the three most represented TE families ( rover , copia and I - element ) with DamID tracks for adult fly intestinal stem cells ( ISC ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B Somatic TE insertion sites of rover LTR elements were depleted from intergenic and exonic sequences and enriched in introns and 3'UTR regions of the fly genome. Insertions of copia LTR elements were also enriched in introns.  D Correlations of somatic insertion sites of the three most represented TE families (rover, copia and I-element) with modENCODE tracks for adult fly tissues.  E Correlations of somatic insertion sites of the three most represented TE families (rover, copia and I-element) with DamID tracks for adult fly intestinal stem cells (ISC).


Figure 1 ( A ) Western blot analysis of Caspase 8 , FLIP and FADD recruitment to the TRAIL - R2 DISC in A549 , HCT116 and DU145 cell lines following incubation with increasing concentrations ( 1x , 2x , or 4x ) of anti - TRAIL - R2 ( AMG655 ) - conjugated magnetic beads for 90 minutes . ( B ) Western blot analysis of the unbound soluble fraction An untreated input of each cell line is included for comparison . ( C ) Quantification of Western blot analysis for FLIP , FADD , Procaspase 8 and their respective cleavage fragments at the TRAIL - R2 DISC . Proteins were quantified by densitometry and normalised to known protein standards ( D ) Caspase - 8 - like ( IETDase ) activity assay in the unbound soluble fraction from the DISC IP An untreated control ( 0 ) was used for each cell line to show basal caspase - 8 activity . ( E ) Ratios of DED proteins quantified at the TRAIL - R2 DISC in A549 , HCT116 and DU145 cells ; increasing concentrations of AMG655 are indicated by colour intensity . ( F ) Correlation between caspase - 8 activity and ratio of FADD : FLIP ( L ) .

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

Figure 1(A) Western blot analysis of Caspase 8, FLIP and FADD recruitment to the TRAIL-R2 DISC in A549, HCT116 and DU145 cell lines following incubation with increasing concentrations (1x, 2x, or 4x) of anti-TRAIL-R2 (AMG655)-conjugated magnetic beads for 90 minutes.(B) Western blot analysis of the unbound soluble fraction An untreated input of each cell line is included for comparison.(C) Quantification of Western blot analysis for FLIP, FADD, Procaspase 8 and their respective cleavage fragments at the TRAIL-R2 DISC. Proteins were quantified by densitometry and normalised to known protein standards(D) Caspase-8-like (IETDase) activity assay in the unbound soluble fraction from the DISC IP An untreated control (0) was used for each cell line to show basal caspase-8 activity.(E) Ratios of DED proteins quantified at the TRAIL-R2 DISC in A549, HCT116 and DU145 cells; increasing concentrations of AMG655 are indicated by colour intensity.(F) Correlation between caspase-8 activity and ratio of FADD:FLIP(L).


Figure 2 ( A ) Western blot analysis of FADD , FLIP and Caspase 8 at the TRAIL - R2 DISC in HCT116 cells treated with escalating doses ( 0 - 30nM ) of caspase - 8 siRNA for 48 hours . Samples were incubated with anti - TRAIL - R2 ( AMG655 ) beads ( 4x ) for 90 minutes prior to collection . A scrambled control siRNA ( SC ) was transfected ( 30nM ) for comparison . ( B ) Quantification of Caspase 8 ( p55 , p43 / 41 and p26 / 24 ) and FLIP ( FLIP ( L ) , FLIP ( S ) and p43 - FLIP ) at the TRAIL - R2 DISC by densitometry and normalised to known protein standards ( C ) Ratio of Caspase - 8 : FLIP calculated from values presented in ( B ) . ( D ) Caspase - 8 ( IETDase ) and - 3 / 7 ( DEVDase ) activity assay in the unbound soluble fraction ( E ) Western blot analysis of FLIP , Caspase 8 , and FADD recruitment to the TRAIL - R2 DISC in PC3 cells stably expressing an empty vector ( EV ) or flag tagged FLIP L ( FL ) treated with 2 . 5µM MS - 275 for 48 hours , followed by a 90 minute DISC IP . * modified form of FLIP , potentially mono - ubiquitinated p43 - FLIP ( L ) . ( G ) Caspase - 8 ( IETDase ) activity assays of the unbound soluble

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

Figure 2(A) Western blot analysis of FADD, FLIP and Caspase 8 at the TRAIL-R2 DISC in HCT116 cells treated with escalating doses (0-30nM) of caspase-8 siRNA for 48 hours. Samples were incubated with anti-TRAIL-R2 (AMG655) beads (4x) for 90 minutes prior to collection. A scrambled control siRNA (SC) was transfected (30nM) for comparison. (B) Quantification of Caspase 8 (p55, p43/41 and p26/24) and FLIP (FLIP(L), FLIP(S) and p43-FLIP) at the TRAIL-R2 DISC by densitometry and normalised to known protein standards (C) Ratio of Caspase-8:FLIP calculated from values presented in (B).(D) Caspase-8 (IETDase) and -3/7 (DEVDase) activity assay in the unbound soluble fraction(E) Western blot analysis of FLIP, Caspase 8, and FADD recruitment to the TRAIL-R2 DISC in PC3 cells stably expressing an empty vector (EV) or flag tagged FLIP L (FL) treated with 2.5µM MS-275 for 48 hours, followed by a 90 minute DISC IP. * modified form of FLIP, potentially mono-ubiquitinated p43-FLIP(L).(G) Caspase-8 (IETDase) activity assays of the unbound soluble fraction


Figure 3 ( A ) Western Blot analysis of Caspase 8 , Caspase 10 , FLIP and FADD recruitment to the TRAIL - R2 DISC in procaspase 8 ( WT ) or procaspase 8 deficient ( Null ) A549 cells incubated with AMG655 beads ( 4x ) for either 30 , 60 or 180 minutes . ( B ) Western blot analysis of the recruitment of FLIP , Caspase 8 , Caspase 10 and FADD to the TRAIL - R2 DISC in HCT116 Parental , caspase 8 null ( CASP8 ) , caspase 10 null ( CASP10 ) or CASP 8 / 10 cells treated with 4x AMG655 conjugated beads for 90 minutes . ( C ) AlphaScreen ® assessment of the interaction of recombinant FLIP DED1 / 2 and recombinant FADD DED . ( D ) AlphaScreen ® assessment of the impact on the FLIP - FADD interaction of unstapled and stapled peptides corresponding to the FADD α1 helix . ( E ) NanoBiT ® assay to quantify FLIP / FADD interactions in a U2OS cell line stably expressing the NanoBiT ® constructs , FLIP ( LgBiT ) and FADD ( smBiT ) following 48 hour silencing of FLIP , FADD , or caspase 8 . Western blot analysis was used to confirm successful knockdown of target proteins by siRNA . ( F ) NanoBiT ® assay of HCT116 and U20S wild type ( WT ) and procaspase 8 null ( Null ) cells transiently co - transfected with FLIP ( LgBiT ) and FADD ( SmBiT ) NanoBiT ® constructs for 24 hours . Construct expression was analysed by Western blotting .

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

Figure 3(A) Western Blot analysis of Caspase 8, Caspase 10, FLIP and FADD recruitment to the TRAIL-R2 DISC in procaspase 8 (WT) or procaspase 8 deficient (Null) A549 cells incubated with AMG655 beads (4x) for either 30, 60 or 180 minutes.(B) Western blot analysis of the recruitment of FLIP, Caspase 8, Caspase 10 and FADD to the TRAIL-R2 DISC in HCT116 Parental, caspase 8 null (CASP8), caspase 10 null (CASP10) or CASP 8/10 cells treated with 4x AMG655 conjugated beads for 90 minutes.(C) AlphaScreen® assessment of the interaction of recombinant FLIP DED1/2 and recombinant FADD DED.(D) AlphaScreen® assessment of the impact on the FLIP-FADD interaction of unstapled and stapled peptides corresponding to the FADD α1 helix.(E) NanoBiT® assay to quantify FLIP/FADD interactions in a U2OS cell line stably expressing the NanoBiT® constructs, FLIP (LgBiT) and FADD (smBiT) following 48 hour silencing of FLIP, FADD, or caspase 8. Western blot analysis was used to confirm successful knockdown of target proteins by siRNA.(F) NanoBiT® assay of HCT116 and U20S wild type (WT) and procaspase 8 null (Null) cells transiently co-transfected with FLIP (LgBiT) and FADD (SmBiT) NanoBiT® constructs for 24 hours. Construct expression was analysed by Western blotting.


Figure 4 ( A ) Western blot analysis of FLIP , Caspase 8 and FADD recruitment to the TRAIL - R2 DISC in U20S parental cells ( EV ) and 3 independent caspase 8 null clones ( # 1 , # 2 , # 3 ) after incubation with AMG655 conjugated beads for 30 , 60 or 180 minutes . ( B ) Western blot analysis of FLIP , Caspase 8 and FADD in the soluble unbound fraction ( C ) Quantification of FADD recruitment to the TRAIL - R2 DISCs FADD levels were normalised to TRAIL - R2 levels in the pull - downs . Densitometry was performed using ImageJ ® . ( E ) Western blot analysis of FADD recruitment to the TRAIL - R2 DISC in HCT116 cells transiently transfected with an empty vector ( EV ) , Flag - tagged FADD ( WT ) , or Flag tagged FADD with point mutations at either Phe25 ( F25A ) or His9 ( H9G ) . Western blotting analysis of the soluble unbound fraction was used to monitor transfection efficiency . ( F ) NanoBiT ® assay of U20S cells transiently co - transfected with caspase 8 ( LgBiT ) and either WT FADD smBiT or FADD ( smBiT ) constructs with a point mutation at either phenylalanine 25 ( F25A ) or histidine 9 ( H9G ) for 48 hours . Western blot analysis was used to assess the expression level of each construct .

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Figure 4(A) Western blot analysis of FLIP, Caspase 8 and FADD recruitment to the TRAIL-R2 DISC in U20S parental cells (EV) and 3 independent caspase 8 null clones (#1, #2, #3) after incubation with AMG655 conjugated beads for 30, 60 or 180 minutes.(B) Western blot analysis of FLIP, Caspase 8 and FADD in the soluble unbound fraction(C) Quantification of FADD recruitment to the TRAIL-R2 DISCs FADD levels were normalised to TRAIL-R2 levels in the pull-downs. Densitometry was performed using ImageJ®.(E) Western blot analysis of FADD recruitment to the TRAIL-R2 DISC in HCT116 cells transiently transfected with an empty vector (EV), Flag-tagged FADD (WT), or Flag tagged FADD with point mutations at either Phe25 (F25A) or His9 (H9G). Western blotting analysis of the soluble unbound fraction was used to monitor transfection efficiency.(F) NanoBiT® assay of U20S cells transiently co-transfected with caspase 8 (LgBiT) and either WT FADD smBiT or FADD (smBiT) constructs with a point mutation at either phenylalanine 25 (F25A) or histidine 9 (H9G) for 48 hours. Western blot analysis was used to assess the expression level of each construct.


Figure 5 ( A ) Western blot analysis of FLIP , caspase 8 and FADD recruitment to the TRAIL - R2 DISC in parental ( WT ) and CFLAR deficient ( KO ) HAP - 1 cells treated with AMG655 conjugated beads for either 45 minutes ( 45 ' ) , 90 minutes ( 90 ' ) , 180 minutes ( 180 ' ) , 3 hours ( 3h ) , 6 hours ( 6h ) or 9 hours ( 9h ) . ( B ) Caspase activity assays of the unbound soluble fraction ( C ) Caspase - 8 ( i ) and - 3 / 7 ( ii ) activity assays and high content microscopy assessment of cell death ( iii ) in Parental ( PAR ) and FLIP knockout ( CFLAR CRISPR ) cells treated with escalating doses of isoleucine zipper TRAIL ( IZ - TRAIL ) for 6 hours . ( D ) Caspase - 8 ( i ) and - 3 / 7 ( ii ) activity assays in Parental ( PAR ) and FLIP knockout ( CFLAR CRISPR ) cells treated with escalating doses of isoleucine zipper TRAIL ( IZ - TRAIL ) for 24 hours . ( iii ) Flow cytometry assessment of cell death induction in Parental ( PAR ) and FLIP knockout ( CFLAR CRISPR ) cells treated with IZ - TRAIL for 24h . ( E ) Western Blot analysis of Parental ( WT ) and FLIP knockout ( KO ) HAP - 1 cells treated with 10ng / mL IZ - TRAIL for 0 , 3 , 6 or 24 hours . ( F ) Cell viability assay of Parental ( PAR ) and FLIP knockout ( CFLAR CRISPR cells ) treated with an escalating dose ( 0 - 1000ng / mL ) of IZ - TRAIL for 72 hours .

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Figure 5(A) Western blot analysis of FLIP, caspase 8 and FADD recruitment to the TRAIL-R2 DISC in parental (WT) and CFLAR deficient (KO) HAP-1 cells treated with AMG655 conjugated beads for either 45 minutes (45'), 90 minutes (90'), 180 minutes (180'), 3 hours (3h), 6 hours (6h) or 9 hours (9h).(B) Caspase activity assays of the unbound soluble fraction(C) Caspase-8 (i) and -3/7 (ii) activity assays and high content microscopy assessment of cell death (iii) in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 6 hours.(D) Caspase-8 (i) and -3/7 (ii) activity assays in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 24 hours. (iii) Flow cytometry assessment of cell death induction in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with IZ-TRAIL for 24h.(E) Western Blot analysis of Parental (WT) and FLIP knockout (KO) HAP-1 cells treated with 10ng/mL IZ-TRAIL for 0, 3, 6 or 24 hours.(F) Cell viability assay of Parental (PAR) and FLIP knockout (CFLAR CRISPR cells) treated with an escalating dose (0-1000ng/mL) of IZ-TRAIL for 72 hours.


Figure 6 ( B ) NanoBiT ® assay performed on TRAIL - R2 DISC IPs ( 90 ' ) carried out in U20S Caspase - 8 null cells co - transfected with wild - type or F114A FLIP - LgBiT fusion proteins and wild - type or F122A caspase - 8 - SmBiT fusion proteins . ( E ) Western blot analysis of size exclusion chromatography experiments in which HCT116 cells transfected with Flag - tagged wild - type ( WT ) FLIP ( S ) were treated with 100ng / mL IZ - TRAIL for 3h prior to cell lysis and separation of low and high MW complexes using a size exclusion column . The fractions eluted at 2MDa and > 2MDa are presented .

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Figure 6(B) NanoBiT® assay performed on TRAIL-R2 DISC IPs (90') carried out in U20S Caspase-8 null cells co-transfected with wild-type or F114A FLIP-LgBiT fusion proteins and wild-type or F122A caspase-8-SmBiT fusion proteins.(E) Western blot analysis of size exclusion chromatography experiments in which HCT116 cells transfected with Flag-tagged wild-type (WT) FLIP(S) were treated with 100ng/mL IZ-TRAIL for 3h prior to cell lysis and separation of low and high MW complexes using a size exclusion column. The fractions eluted at 2MDa and >2MDa are presented.


Figure 2IE were isolated and binding to HBMEC and HDMEC was determined under flow conditions . Number of IE bound per mm2 EC surface was calculated and shown is the mean ± 95 % CI for 26 CM and 33 UM cases on HBMEC and 21 CM and 35 UM cases on HDMEC on a log scale . P - value was calculated by two - tailed unpaired t - test . The dotted line is 20 IE / mm2 , the cut - off value for inclusion of the inhibition data .

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Figure 2IE were isolated and binding to HBMEC and HDMEC was determined under flow conditions. Number of IE bound per mm2 EC surface was calculated and shown is the mean ± 95% CI for 26 CM and 33 UM cases on HBMEC and 21 CM and 35 UM cases on HDMEC on a log scale. P-value was calculated by two-tailed unpaired t-test. The dotted line is 20 IE/mm2, the cut-off value for inclusion of the inhibition data.


Figure 3IE were isolated and binding to HBMEC and HDMEC was determined under flow conditions in the absence and presence of 5 µg / ml αICAM - 1 or αCD36 antibody or 50 µg / ml rEPCR . Number of IE bound per mm2 EC surface was determined . IE were isolated and binding to HBMEC and HDMEC was determined under flow conditions in the absence and presence of 5 µg / ml αICAM - 1 or αCD36 antibody or 50 µg / ml rEPCR . Number of IE bound per mm2 EC surface was determined . Using the same data , percentage inhibition by αICAM - 1 antibody was calculated relatively to binding in the absence of antibody . Using the same data , percentage inhibition by rEPCR was calculated relatively to binding in the absence of inhibitor . Using the same data , percentage inhibition by αCD36 antibody was calculated relatively to binding in the absence of antibody .

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Figure 3IE were isolated and binding to HBMEC and HDMEC was determined under flow conditions in the absence and presence of 5 µg/ml αICAM-1 or αCD36 antibody or 50 µg/ml rEPCR. Number of IE bound per mm2 EC surface was determined.IE were isolated and binding to HBMEC and HDMEC was determined under flow conditions in the absence and presence of 5 µg/ml αICAM-1 or αCD36 antibody or 50 µg/ml rEPCR. Number of IE bound per mm2 EC surface was determined. Using the same data, percentage inhibition by αICAM-1 antibody was calculated relatively to binding in the absence of antibody. Using the same data, percentage inhibition by rEPCR was calculated relatively to binding in the absence of inhibitor. Using the same data, percentage inhibition by αCD36 antibody was calculated relatively to binding in the absence of antibody.


Figure 1 ( 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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Figure 1(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.


Figure 2 ( 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 . ( D ) Super - enhancer identification . Tissue - specific enhancers are identified , so that they are listed more than twice ( twice : blue , all : red ) in the top 5 % in all enhancer candidates and are not in the SEs of other tissues . Grey shades indicate the top 5 % of tag count in enhancer candidates . ( F ) Hnf4ɑ binds to the SE at Alb gene loci .

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Figure 2(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.(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.


Figure 3 ( 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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Figure 3(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.


Figure 4 ( 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 . ( 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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Figure 4(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.(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.


Figure 5 ( 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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Figure 5(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).


Figure 2 GSEA analysis showing enrichment of BCC [ 28 ] , well - differentiated and EMT containing SCC [ 33 ] for YAP gene signatures obtained from mouse hepatocellular carcinoma [ 40 ] human breast cancer cell line [ 39 ] , human oral squamous cell carcinoma [ 42 ] and human colorectal cancer [ 41 ] . Well - diff SCC : well - differentiated SCC ; EMT SCC : EMT - containing SCC

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Figure 2 GSEA analysis showing enrichment of BCC [28], well-differentiated and EMT containing SCC [33] for YAP gene signatures obtained from mouse hepatocellular carcinoma [40]human breast cancer cell line [39], human oral squamous cell carcinoma [42] and human colorectal cancer [41]. Well-diff SCC: well-differentiated SCC; EMT SCC: EMT-containing SCC