Shown is one representative Western blot from two independent experiments and quantification of LC3 - II / I and p62 protein levels in siCtrl ( n = 4 biological replicates ) or siASK1 ( n = 4 biological replicates ) cells treated with Palm .

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Shown is one representative Western blot from two independent experiments and quantification of LC3-II/I and p62 protein levels in siCtrl (n=4 biological replicates) or siASK1 (n=4 biological replicates) cells treated with Palm.


Colocalization of LC3 - II punctate ( red ; arrow ) with lipid ( BODIPY 493 / 503 , green ) and nuclei ( Hoechst , blue ) in siCtrl or siASK1 cells treated with BSA , Palm or Palm + Rap . Scale bar represents 100 µm .

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Colocalization of LC3-II punctate (red; arrow) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in siCtrl or siASK1 cells treated with BSA, Palm or Palm+Rap. Scale bar represents 100 µm.


Colocalization of LC3 - II punctate with lipid droplets ( BODIPY 493 / 503 , green ) was quantified in hepatocytes transfected with siRNA targeting ASK1 ( siASK1 ; grey bars ) or non - targeting siRNA control ( siCtrl ; black bars ) and treated with BSA or BSA + Baf for 24 hours ( n = 4 biological replicates ) .

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Colocalization of LC3-II punctate with lipid droplets (BODIPY 493/503, green) was quantified in hepatocytes transfected with siRNA targeting ASK1 (siASK1; grey bars) or non-targeting siRNA control (siCtrl; black bars) and treated with BSA or BSA+Baf for 24 hours (n=4 biological replicates).


Lipid accumulation was quantified using automated image - based analysis in ASK1 knockdown ( siASK1 ; grey bars ) or control ( siCtrl ; black bars ) cells treated with BSA , Palm or Palm + Baf and stained as mentioned above ( n = 4 biological replicates ) . Values are expressed as mean ± SEM . *

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Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm or Palm+Baf and stained as mentioned above (n=4 biological replicates). Values are expressed as mean ± SEM. *


Representative images of liver sections stained with H & E from mice fed a HFD for 20 weeks . Scale bar represents 100 µm .

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Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.


Liver triglyceride ( TG ) content of mice fed a chow ( ASK1F / F n = 6 mice ; ASK1Δhep n = 6 mice ) or HFD ( ASK1F / F n = 14 mice ; ASK1Δhep n = 15 mice ) .

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Liver triglyceride (TG) content of mice fed a chow (ASK1F/F n=6 mice; ASK1Δhep n=6 mice) or HFD (ASK1F/F n=14 mice; ASK1Δhep n=15 mice).


Relative mRNA expression of perilipin ( Plin ) and very low - density lipoprotein receptor ( Vldlr ) in liver of mice fed a HFD for 20 weeks ( ASK1F / F n = 6 mice ; ASK1Δhep n = 7 mice ( Vldlr ) or n = 8 mice ( Plin ) ) .

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Relative mRNA expression of perilipin (Plin) and very low-density lipoprotein receptor (Vldlr) in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (Vldlr) or n=8 mice (Plin)).


Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks ( ASK1F / F n = 6 mice ; ASK1Δhep n = 7 mice ( F4 / 80 , Mcp1 ) or ASK1Δhep n = 8 mice ( Il - 6 ) ) .

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Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (F4/80, Mcp1) or ASK1Δhep n=8 mice (Il-6)).


Relative mRNA expression of collagen , type I , alpha 1 ( Col1a1 ) ( ASK1F / F n = 11 mice ; ASK1Δhep n = 15 mice ) and transforming growth factor beta 1 ( Tgfβ1 ) ( ASK1F / F n = 6 mice ; ASK1Δhep n = 8 mice ) in liver of HFD - fed mice .

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Relative mRNA expression of collagen, type I, alpha 1 (Col1a1) (ASK1F/F n=11 mice; ASK1Δhep n=15 mice) and transforming growth factor beta 1 (Tgfβ1) (ASK1F/F n=6 mice; ASK1Δhep n=8 mice) in liver of HFD-fed mice.


Representative images of liver sections stained with Sirius Red ( scale bar represents 100 µm )

Representative images of liver sections stained with Sirius Red (scale bar represents 100 µm)


quantification of Sirius Red positive area in liver of mice fed a HFD ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) .

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quantification of Sirius Red positive area in liver of mice fed a HFD (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).


Representative images of liver sections stained with α - SMA from mice fed a HFD for 20 weeks . Scale bar represents 100 µm .

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Representative images of liver sections stained with α-SMA from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.


Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) .

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Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).


Liver triglyceride ( TG ) content ( ASK1F / F n = 7 mice ; ASK1Δhep n = 7 mice ) and quantification of Sirius Red positive area in livers ( ASK1F / F n = 7 mice ; ASK1Δhep n = 7 mice ) of 15 months old chow - fed mice . Values are expressed as mean ± SEM . #

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Liver triglyceride (TG) content (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) and quantification of Sirius Red positive area in livers (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) of 15 months old chow-fed mice. Values are expressed as mean ± SEM. #


Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks ( ASK1F / F n = 8 mice ; ASK1Δhep n = 8 mice ) .

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Shown is one representative Western blots from two independent experiments and quantification of respective protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).


Representative immunofluorescence images of liver sections stained for LC3 - II punctate ( red ) and nuclei ( DAPI , blue ) in mice . Scale bar represents 100 µm .

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Representative immunofluorescence images of liver sections stained for LC3-II punctate (red) and nuclei (DAPI, blue) in mice. Scale bar represents 100 µm.


Primary hepatocytes isolated from chow - fed ASK1F / F ( ) and ASK1Δhep ( ) mice were treated with BSA , Palm , Palm + Rap or Palm + Rap + Baf for 24 h . Cells were stained for lipid droplet accumulation ( Bodipy 493 / 503 , green ) and nuclei ( Hoechst , blue ) . Lipid accumulation was quantified using automated image - based analysis ( n = 4 biological replicates ) .

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Primary hepatocytes isolated from chow-fed ASK1F/F () and ASK1Δhep () mice were treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates).


Colocalization of LC3 - II punctate ( red ) with lipid ( BODIPY 493 / 503 , green ) and nuclei ( Hoechst , blue ) in primary hepatocytes treated with BSA , Palm , Palm + Rap or Palm + Rap + Baf . Scale bar represents 100 µm . Values are expressed as mean ± SEM .

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Colocalization of LC3-II punctate (red) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in primary hepatocytes treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf. Scale bar represents 100 µm. Values are expressed as mean ± SEM.


Western blots and quantification of respective protein levels in liver of HFD - fed mice treated with or without ASK1 inhibitor ( n = 4 mice per treatment ) .

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Western blots and quantification of respective protein levels in liver of HFD-fed mice treated with or without ASK1 inhibitor (n=4 mice per treatment).


Representative images of liver sections stained with H & E ( scale bar represents 100 µm )

Representative images of liver sections stained with H&E (scale bar represents 100 µm)


liver triglyceride ( TG ) content of HFD - fed mice treated with or without ASK1 inhibitor ( n = 8 mice per treatment ) .

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liver triglyceride (TG) content of HFD-fed mice treated with or without ASK1 inhibitor (n=8 mice per treatment).


Representative images of liver sections stained with H & E from mice fed a HFD for 20 weeks . Scale bar represents 100 µm .

Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm.


Liver triglyceride ( TG ) content ( ASK1F / F n = 11 mice ; ASK1 + hep n = 10 mice ) in livers of mice fed a HFD for 20 weeks .

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Liver triglyceride (TG) content (ASK1F/F n=11 mice; ASK1+hep n=10 mice) in livers of mice fed a HFD for 20 weeks.


Western blots and quantification of protein levels of respective targets in liver of HFD - fed mice ( n = 8 mice per genotype for pJNK and ATG12 , n = 4 mice per genotype for pBCN1 ) .

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Western blots and quantification of protein levels of respective targets in liver of HFD-fed mice (n=8 mice per genotype for pJNK and ATG12, n=4 mice per genotype for pBCN1).


Quantification of Sirius Red positive area ( ASK1F / F n = 11 mice ; ASK1 + hep n = 9 mice ) in livers of CCl4 - injected mice .

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Quantification of Sirius Red positive area (ASK1F/F n=11 mice; ASK1+hep n=9 mice) in livers of CCl4-injected mice.


Relative mRNA expression ( ASK1F / F n = 13 mice ; ASK1 + hep n = 14 mice ) of respective genes involved in inflammation and fibrosis in liver of CCl4 - injected mice . Values are expressed as mean ± SEM . #

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Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM. #


Relative mRNA expression ( ASK1F / F n = 13 mice ; ASK1 + hep n = 14 mice ) of respective genes involved in inflammation and fibrosis in liver of CCl4 - injected mice . Values are expressed as mean ± SEM .

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Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM.


A Whole mount FISH for piwi - 1 and immunostaining for BrdU at 1 - day , 1 - week and 5 - weeks post - BrdU administration . White boxes denote the magnified regions shown in the bottom panels . Scale bars , 30 µm .

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A Whole mount FISH for piwi-1 and immunostaining for BrdU at 1-day, 1-week and 5-weeks post-BrdU administration. White boxes denote the magnified regions shown in the bottom panels. Scale bars, 30 µm.


B Quantification of the percentage of BrdU label - retaining neoblasts . Data are presented as mean ± s . d . ( n = 4 - 6 animals per time point ) .

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B Quantification of the percentage of BrdU label-retaining neoblasts. Data are presented as mean ± s.d. (n = 4-6 animals per time point).


C Double pulse experiment showing colocalization of BrdU after a 2 - week chase period with EdU after a 1 - day chase period . White boxes denote the magnified region shown on the left . Dashed lines mark the edges of the animal and white arrows mark colocalization . Scale bar , 50 µm .

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C Double pulse experiment showing colocalization of BrdU after a 2-week chase period with EdU after a 1-day chase period. White boxes denote the magnified region shown on the left. Dashed lines mark the edges of the animal and white arrows mark colocalization. Scale bar, 50 µm.


A Dissociated cells from unirradiated , 1 dpi or 7 dpi animals ( 60 Gy of irradiation ) were stained with Hoechst ( DNA ) and Pyronin Y ( RNA ) and sorted by FACS to reveal an RNAlow population . Percentages indicate the proportion of events within each gate .

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A Dissociated cells from unirradiated, 1 dpi or 7 dpi animals (60 Gy of irradiation) were stained with Hoechst (DNA) and Pyronin Y (RNA) and sorted by FACS to reveal an RNAlow population. Percentages indicate the proportion of events within each gate.


B Cell size and nucleus : cytoplasm ratio measured by CellMask ( cytoplasm ) and DAPI ( nucleus ) staining of FACS sorted cells at homeostasis ( n ≥ 47 per sample ) . Representative images are shown , with dashed lines to indicate the area measured . Scale bars , 5 µm .

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B Cell size and nucleus:cytoplasm ratio measured by CellMask (cytoplasm) and DAPI (nucleus) staining of FACS sorted cells at homeostasis (n ≥ 47 per sample). Representative images are shown, with dashed lines to indicate the area measured. Scale bars, 5 µm.


C EdU pulse - chase time course of label - retention in sorted RNAlow and S / G2 / M neoblasts ( n ≥ 3 individual experiments per time point ) . Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S / G2 / M or RNAlow neoblasts , respectively . Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S / G2 / M neoblasts .

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C EdU pulse-chase time course of label-retention in sorted RNAlow and S/G2/M neoblasts (n ≥ 3 individual experiments per time point). Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S/G2/M or RNAlow neoblasts, respectively. Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S/G2/M neoblasts.


D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C . Red boxes denote examples of EdU - cells . Scale bars , 5 µm .

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D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C. Red boxes denote examples of EdU- cells. Scale bars, 5 µm.


A Area measurements of cells from the RNAlow , G1 and S / G2 / M gates in intact animals and at 6 and 48 hpa ( n ≥ 28 for each sample in the RNAlow plot , n ≥ 24 in the G1 plot , n ≥ 42 in the S / G2 / M plot ) .

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A Area measurements of cells from the RNAlow, G1 and S/G2/M gates in intact animals and at 6 and 48 hpa (n ≥ 28 for each sample in the RNAlow plot, n ≥ 24 in the G1 plot, n ≥ 42 in the S/G2/M plot).


B RNAlow neoblast size in intact animals and at 48 , 72 and 96 hpa ( n ≥ 54 ) .

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B RNAlow neoblast size in intact animals and at 48, 72 and 96 hpa (n ≥ 54).


C RNAlow neoblast size in intact animals and at 48 hours following amputation , poke or incision injuries ( n ≥ 61 ) .

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C RNAlow neoblast size in intact animals and at 48 hours following amputation, poke or incision injuries (n ≥ 61).


D RNAlow neoblast size in unirradiated animals and at 1 , 2 and 3 dpi ( 12 . 5 Gy . of irradiation , n ≥ 52 ) .

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D RNAlow neoblast size in unirradiated animals and at 1, 2 and 3 dpi (12.5 Gy. of irradiation, n ≥ 52).


A Representative images of RNAlow , G1 and S / G2 / M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa . All images shown are from samples processed in the same experiment . Intact images are duplicated from Figure 2B . White arrows mark projections . Percentage of cells displaying projections in each condition is noted ( n > 100 cells counted ) . Scale bars , 5 µm . B Average projection length in RNAlow , G1 and S / G2 / M cells from intact animals and at 6 and 48 hpa ( n ≥ 13 ) . C - E Quantification of the number of projections per cell ( blue data ) and the sum total projection length per cell ( orange data ) in RNAlow ( C ) , G1 ( D ) or S / G2 / M ( E ) cells isolated from intact animals and at 6 and 48 hpa ( n ≥ 23 for projections / cell , n ≥ 10 for Σ projection length / cell ) .

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A Representative images of RNAlow, G1 and S/G2/M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa. All images shown are from samples processed in the same experiment. Intact images are duplicated from Figure 2B. White arrows mark projections. Percentage of cells displaying projections in each condition is noted (n > 100 cells counted). Scale bars, 5 µm. B Average projection length in RNAlow, G1 and S/G2/M cells from intact animals and at 6 and 48 hpa (n ≥ 13). C-E Quantification of the number of projections per cell (blue data) and the sum total projection length per cell (orange data) in RNAlow (C), G1 (D) or S/G2/M (E) cells isolated from intact animals and at 6 and 48 hpa (n ≥ 23 for projections/cell, n ≥ 10 for Σ projection length/cell).


A Representative images of RNAlow , G1 and S / G2 / M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa . All images shown are from samples processed in the same experiment . Intact images are duplicated from Figure 2B . White arrows mark projections . Percentage of cells displaying projections in each condition is noted ( n > 100 cells counted ) . Scale bars , 5 µm .

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A Representative images of RNAlow, G1 and S/G2/M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa. All images shown are from samples processed in the same experiment. Intact images are duplicated from Figure 2B. White arrows mark projections. Percentage of cells displaying projections in each condition is noted (n > 100 cells counted). Scale bars, 5 µm.


B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment .

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B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment.


C RNAlow neoblast size in intact animals and at 48 hpa in control or TOR KD animals ( n ≥ 55 ) .

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C RNAlow neoblast size in intact animals and at 48 hpa in control or TOR KD animals (n ≥ 55).


D Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals ( n = 5 - 10 ) .

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D Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals (n = 5-10).


E RNAlow neoblast size in intact animals and at 48 hpa in control , raptor or rictor KD animals ( n ≥ 54 ) .

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E RNAlow neoblast size in intact animals and at 48 hpa in control, raptor or rictor KD animals (n ≥ 54).


F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals . Percentages indicate the proportion of events within each gate .

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F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals. Percentages indicate the proportion of events within each gate.


A Representative WISH images for Lrig - 1 .

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A Representative WISH images for Lrig-1.


B RNAlow neoblast size in intact animals following control or Lrig - 1 knockdown ( n ≥ 53 technical replicates ) .

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B RNAlow neoblast size in intact animals following control or Lrig-1 knockdown (n ≥ 53 technical replicates).


C Quantification of H3P immunostaining in intact animals following control or Lrig - 1 knockdown ( n = 9 - 10 technical replicates ) .

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C Quantification of H3P immunostaining in intact animals following control or Lrig-1 knockdown (n = 9-10 technical replicates).


D Quantification of H3P immunostaining at homeostasis ( fixed at 7 days post - RNAi , white region ) and at 48 hpa ( amputated at 5 days post - RNAi and fixed at 7 days post - RNAi , yellow region ) during the first round of regeneration in control or Lrig - 1 KD animals ( n = 10 - 17 ) .

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D Quantification of H3P immunostaining at homeostasis (fixed at 7 days post-RNAi, white region) and at 48 hpa (amputated at 5 days post-RNAi and fixed at 7 days post-RNAi, yellow region) during the first round of regeneration in control or Lrig-1 KD animals (n = 10-17).


E Quantification of unpigmented blastema area ( as % total tissue area ) in regenerating control or Lrig - 1 KD animals from 2 dpa to 7 dpa during the first round of regeneration ( n = 10 animals per condition per time point ) . The same animals were used for all timepoints . Representative images from 7 dpa are shown . Scale bars , 500 µm .

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

E Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the first round of regeneration (n = 10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.


F Quantification of H3P immunostaining at homeostasis ( fixed at 14 days post - RNAi , white region ) and at 48 hpa ( amputated at 12 days post - RNAi and fixed at 14 days post - RNAi , yellow region ) during a second round of regeneration in control or Lrig - 1 KD animals ( n = 10 - 18 ) .

F Quantification of H3P immunostaining at homeostasis (fixed at 14 days post-RNAi, white region) and at 48 hpa (amputated at 12 days post-RNAi and fixed at 14 days post-RNAi, yellow region) during a second round of regeneration in control or Lrig-1 KD animals (n = 10-18).


G Quantification of unpigmented blastema area ( as % total tissue area ) in regenerating control or Lrig - 1 KD animals from 2 dpa to 7 dpa during the second round of regeneration ( n = 9 - 10 animals per condition per time point ) . The same animals were used for all timepoints . Representative images from 7 dpa are shown . Scale bars , 500 µm .

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

G Quantification of unpigmented blastema area (as % total tissue area) in regenerating control or Lrig-1 KD animals from 2 dpa to 7 dpa during the second round of regeneration (n = 9-10 animals per condition per time point). The same animals were used for all timepoints. Representative images from 7 dpa are shown. Scale bars, 500 µm.


A ) Transcriptome comparison of hemocytes from stage 16 embryos ( E16 ) and wandering 3rd instar larvae ( WL ) . The x - axis is the average genes expression levels ( n = 3 ) and the y - axis the Log2 Fold Change WL / E16 . P - values are indicated with the color code .

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

A) Transcriptome comparison of hemocytes from stage 16 embryos (E16) and wandering 3rd instar larvae (WL). The x-axis is the average genes expression levels (n=3) and the y-axis the Log2 Fold Change WL/E16. P-values are indicated with the color code.


Scatter plots as in ( A ) highlighting in black subsets of known genes expressed in hemocytes

Scatter plots as in (A) highlighting in black subsets of known genes expressed in hemocytes


Scatter plots as in ( A ) highlighting in black subsets of known genes expressed in


Scatter plots as in ( A ) highlighting in black subsets of known genes associated with the GO terms extracellular matrix

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

Scatter plots as in (A) highlighting in black subsets of known genes associated with the GO terms extracellular matrix


Scatter plots as in ( A ) highlighting in black subsets of known genes associated with the GO terms extracellular


Scatter plots as in ( A ) highlighting in black subsets of known genes expressed in phagocytosis

Scatter plots as in (A) highlighting in black subsets of known genes expressed in phagocytosis


F - G ) Phagocytosis assay on E16 ( F ) and WL hemocytes ( G ) srp ( hemo ) - moesin - RFP . The beads ( in green ) are phagocytosed by the hemocytes ( in red ) . The WL hemocytes show greater phagocytic capacity compared to the embryonic ones after 5 min of exposure . Full stacks are displayed and the scale bars represent 20 µm .

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

F-G) Phagocytosis assay on E16 (F) and WL hemocytes (G) srp(hemo)-moesin-RFP. The beads (in green) are phagocytosed by the hemocytes (in red). The WL hemocytes show greater phagocytic capacity compared to the embryonic ones after 5 min of exposure. Full stacks are displayed and the scale bars represent 20 µm.


A ) UMAP projection representing the 14 clusters of cells identified in the hemocyte pools from OregonR WL ( NI dataset ) .

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

A) UMAP projection representing the 14 clusters of cells identified in the hemocyte pools from OregonR WL (NI dataset).


D ) Top 5 markers of each cluster . The expression levels is represented by the gradient of purple levels ) and the percentage of cells with the size of the dots .

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

D) Top 5 markers of each cluster. The expression levels is represented by the gradient of purple levels) and the percentage of cells with the size of the dots.


A ) Identification of the position ( circulating / resident ) of the clusters within the larva by qPCR . The left panel indicates the distribution of each marker across all clusters ( as in Figure 2D ) , the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment . Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment ( n = 5 , mean + / - SD are represented on the graph ) . The p - values are estimated by bilateral paired student test

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

A) Identification of the position (circulating/resident) of the clusters within the larva by qPCR. The left panel indicates the distribution of each marker across all clusters (as in Figure 2D), the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment. Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment (n=5, mean +/- SD are represented on the graph). The p-values are estimated by bilateral paired student test


A ) Identification of the position ( circulating / resident ) of the clusters within the larva by qPCR . The left panel indicates the distribution of each marker across all clusters ( as in Figure 2D ) , the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment . Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment ( n = 5 , mean + / - SD are represented on the graph ) . The p - values are estimated by bilateral paired student

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) Circulating and resident hemocytes ( top and bottom panels , respectively ) from Mimic - Lsp1beta - MI05460 WL , which express Lsp1beta tagged with GFP . The immunolabelling was done with anti - GFP ( in gray ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI and GFP , the right panels show the GFP alone . The scale bars represent 10 µm . C ) Quantification of the GFP intensity in circulating and resident hemocytes from MiMIC - Lsp1beta - MI05460 larvae . The OregonR value indicates the background level . The number of cells included in the analysis is displayed on the x - axis label and were quantified from two independent preparation , p - values were estimated after variance analysis with bilateral student test for equal variance .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Circulating and resident hemocytes (top and bottom panels, respectively) from Mimic-Lsp1beta-MI05460 WL, which express Lsp1beta tagged with GFP. The immunolabelling was done with anti-GFP (in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI and GFP, the right panels show the GFP alone. The scale bars represent 10 µm. C) Quantification of the GFP intensity in circulating and resident hemocytes from MiMIC-Lsp1beta-MI05460 larvae. The OregonR value indicates the background level. The number of cells included in the analysis is displayed on the x-axis label and were quantified from two independent preparation, p-values were estimated after variance analysis with bilateral student test for equal variance.


B ) Circulating and resident hemocytes ( top and bottom panels , respectively ) from Mimic - Lsp1beta - MI05460 WL , which express Lsp1beta tagged with GFP . The immunolabelling was done with anti - GFP ( in gray ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI and GFP , the right panels show the GFP alone . The scale bars represent 10 µm . C ) Quantification of the GFP intensity in circulating and resident

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Circulating and resident hemocytes (top and bottom panels, respectively) from Mimic-Lsp1beta-MI05460 WL, which express Lsp1beta tagged with GFP. The immunolabelling was done with anti-GFP (in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI and GFP, the right panels show the GFP alone. The scale bars represent 10 µm. C) Quantification of the GFP intensity in circulating and resident hemocytes


D ) Resident hemocytes located around the oenocytes or along the muscles ( top and bottom panels , respectively ) from WL srp ( hemo ) - moesin - RFP , that express moesin tagged with RFP in hemocytes . The immunolabelling was done with anti - RFP ( in red ) , anti - Pendulin ( Pen , in gray ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI , Pen and RFP , the middle panels RFP alone and the right panels Pen alone . Arrow heads in the top panels indicate cells co - expressing RFP and Pen . The scale bars represent 50 µm .

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

D) Resident hemocytes located around the oenocytes or along the muscles (top and bottom panels, respectively) from WL srp(hemo)-moesin-RFP, that express moesin tagged with RFP in hemocytes. The immunolabelling was done with anti-RFP (in red), anti-Pendulin (Pen, in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, Pen and RFP, the middle panels RFP alone and the right panels Pen alone. Arrow heads in the top panels indicate cells co-expressing RFP and Pen. The scale bars represent 50 µm.


A ) Heatmap representing the z - score for the top 5 regulons of each cluster determined with SCENIC . The dendrogram on the left side of the panel indicates the correlation between the regulons across the dataset . The z - score is indicated with a gradient from magenta ( z - score < 0 , the regulon is repressed ) to yellow ( z - score > 0 , the regulon is active ) .

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

A) Heatmap representing the z-score for the top 5 regulons of each cluster determined with SCENIC. The dendrogram on the left side of the panel indicates the correlation between the regulons across the dataset. The z-score is indicated with a gradient from magenta (z-score < 0, the regulon is repressed) to yellow (z-score > 0, the regulon is active).


A ) UMAP projection representing the 16 clusters of cells identified in the WI sample . Note the presence of two additional clusters , LMLM - 1 and LM - 2 , compared to the NI sample . The two clusters correspond to the lamellocytes .

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

A) UMAP projection representing the 16 clusters of cells identified in the WI sample. Note the presence of two additional clusters, LMLM-1 and LM-2, compared to the NI sample. The two clusters correspond to the lamellocytes.


B ) Estimation of the number of cells of each cluster per larva in normal condition ( NI , orange ) and after wasp infestation ( WI , blue ) . For each cluster , the cell number was deduced from the total number of plasmatocytes and lamellocytes numerated and the proportion of each cluster in the singe cell datasets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Estimation of the number of cells of each cluster per larva in normal condition (NI, orange) and after wasp infestation (WI, blue). For each cluster, the cell number was deduced from the total number of plasmatocytes and lamellocytes numerated and the proportion of each cluster in the singe cell datasets.


D ) Dot plot representing the expression levels ( gradient of red levels ) and the percentage of cells ( size of the dots ) that express the top 5 markers of each cluster in the WI dataset .

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

D) Dot plot representing the expression levels (gradient of red levels) and the percentage of cells (size of the dots) that express the top 5 markers of each cluster in the WI dataset.


A ) Scatter plot comparing the transcriptome of the two lamellocyte clusters , deduced from the WI dataset . The x - axis is the average expression levels of the genes and the y - axis the log2 fold change ( LM - 1 / LM - 2 ) . The genes significantly enriched in the LM - 1 cluster are highlighted in red ( p < 10 - 6 ) , those significantly enriched in the LM - 2 cluster in orange ( p < 10 - 6 ) .

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

A) Scatter plot comparing the transcriptome of the two lamellocyte clusters, deduced from the WI dataset. The x-axis is the average expression levels of the genes and the y-axis the log2 fold change (LM-1/LM-2). The genes significantly enriched in the LM-1 cluster are highlighted in red (p<10-6), those significantly enriched in the LM-2 cluster in orange (p<10-6).


C ) Expression levels ( gradient of color ) and the percentage of cells ( size of the dots ) for the top 10 markers of lamellocytes ( in black ) , of markers specific to LM - 1 compared to LM - 2 ( in red ) and specific to LM - 2 compared to LM - 1 ( in orange ) . Note that most markers enriched in LM - 1 compared to LM - 2 are exclusively expressed in LM - 1 while most markers enriched in LM - 2 compared to LM - 1 are also expressed in the plasmatocyte and crystal cell clusters .

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

C) Expression levels (gradient of color) and the percentage of cells (size of the dots) for the top 10 markers of lamellocytes (in black), of markers specific to LM-1 compared to LM-2 (in red) and specific to LM-2 compared to LM-1 (in orange). Note that most markers enriched in LM-1 compared to LM-2 are exclusively expressed in LM-1 while most markers enriched in LM-2 compared to LM-1 are also expressed in the plasmatocyte and crystal cell clusters.


D ) Hemocytes from WL OregonR infested by wasp . The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea ( in gray , top panels ) and Shot ( in gray , lower panels ) . Phalloidin - FITC ( in green ) labels the actin filament particularly abundant in lamellocytes ( Tokusumi et al . , 2009 ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI , FITC and the lamellocyte markers , the middle panels show the FITC alone and the right panels the lamellocyte markers alone . The scale bars represent 20µm .

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

D) Hemocytes from WL OregonR infested by wasp. The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea (in gray, top panels) and Shot (in gray, lower panels). Phalloidin-FITC (in green) labels the actin filament particularly abundant in lamellocytes (Tokusumi et al., 2009) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, FITC and the lamellocyte markers, the middle panels show the FITC alone and the right panels the lamellocyte markers alone. The scale bars represent 20µm.


A ) Expression levels of the cluster markers in the hemolymph of WI larvae compared to NI larvae . The left panel indicates the distribution of each marker across all clusters ( as in Figure 2D ) , the graph on the right panel , indicates the Log2 of the ratio between the expression level in WI versus NI . Positive values indicate an up - regulation upon wasp infestation and negative values a down - regulation ( n = 3 , pool of 10 larvae per replicate , mean + / - SD are represented on the graph ) . The p - value are estimated by bilateral student test and indicated as follow : ns = not - significant ( > 0 . 05 ) , " * " = p [ 0 . 05 - 0 . 01 [ , " * * " = p [ 0 . 01 - 0 . 001 [ .

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

A) Expression levels of the cluster markers in the hemolymph of WI larvae compared to NI larvae. The left panel indicates the distribution of each marker across all clusters (as in Figure 2D), the graph on the right panel, indicates the Log2 of the ratio between the expression level in WI versus NI. Positive values indicate an up-regulation upon wasp infestation and negative values a down-regulation (n=3, pool of 10 larvae per replicate, mean +/- SD are represented on the graph). The p-value are estimated by bilateral student test and indicated as follow: ns = not-significant (>0.05), "*" = p[0.05 - 0.01[, "**" = p[0.01-0.001[.


B , C ) Expression levels of the cluster markers increasing in WI compared to NI according to ( A ) during the progression of the immune response to wasp infestation . Collections were carried out at t0 ( time of infestation , L2 ) , 24 h ( early L3 ) , 48 h ( mid L3 ) and 72 h ( WL ) after wasp infestation ( n = 3 ) . The time line is on the x - axis and the normalised expression levels on the y - axis . The expression levels were normalised to the levels at t0 . The dotted lines indicate t0 relative level ( = 1 ) . The markers that do not display strong variability during the timeline are presented in ( B ) and the ones showing a strong up - regulation are in ( C ) . D ) Expression levels during the infestation timeline as in ( B , C ) for the markers of proliferation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,C) Expression levels of the cluster markers increasing in WI compared to NI according to (A) during the progression of the immune response to wasp infestation. Collections were carried out at t0 (time of infestation, L2), 24 h (early L3), 48 h (mid L3) and 72 h (WL) after wasp infestation (n=3). The time line is on the x-axis and the normalised expression levels on the y-axis. The expression levels were normalised to the levels at t0. The dotted lines indicate t0 relative level (=1). The markers that do not display strong variability during the timeline are presented in (B) and the ones showing a strong up-regulation are in (C). D) Expression levels during the infestation timeline as in (B,C) for the markers of proliferation.


B , C ) Expression levels of the cluster markers increasing in WI compared to NI according to ( A ) during the progression of the immune response to wasp infestation . Collections were carried out at t0 ( time of infestation , L2 ) , 24 h ( early L3 ) , 48 h ( mid L3 ) and 72 h ( WL ) after wasp infestation ( n = 3 ) . The time line is on the x - axis and the normalised expression levels on the y - axis . The expression levels were normalised to the levels at t0 . The dotted lines indicate t0 relative level ( = 1 ) . The markers that do not display strong variability during the timeline are presented in ( B ) and the ones showing a strong up - regulation are in ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,C) Expression levels of the cluster markers increasing in WI compared to NI according to (A) during the progression of the immune response to wasp infestation. Collections were carried out at t0 (time of infestation, L2), 24 h (early L3), 48 h (mid L3) and 72 h (WL) after wasp infestation (n=3). The time line is on the x-axis and the normalised expression levels on the y-axis. The expression levels were normalised to the levels at t0. The dotted lines indicate t0 relative level (=1). The markers that do not display strong variability during the timeline are presented in (B) and the ones showing a strong up-regulation are in (C).


E ) Weighted dot plot representing the enrichment ( Log2 fold change ) of the markers in hemocytes originating from the lymph gland ( DotGFP - WI ) compared to embryonic hemocytes ( srpGFP - WI ) from WI larva . The lymph gland hemocytes and embryonic hemocytes were traced using the lymph gland driver DotGal4 and the embryonic driver Srp ( hemo ) Gal4 respectively combined with a lineage tracing construct . The relative expression levels are indicated by the size of the dots and the pvalue of the log2 fold change with the gradient dark blue ( not significant ) to light blue ( p < 10 - 3 ) . The p - value are estimated by bilateral student test and indicated as follow : ns = not - significant ( > 0 . 05 ) , " * " = p [ 0 . 05 - 0 . 01 [ , " * * " = p [ 0 . 01 - 0 . 001 [ , " * * * " = p < 0 . 001 ] .

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

E) Weighted dot plot representing the enrichment (Log2 fold change) of the markers in hemocytes originating from the lymph gland (DotGFP-WI) compared to embryonic hemocytes (srpGFP-WI) from WI larva. The lymph gland hemocytes and embryonic hemocytes were traced using the lymph gland driver DotGal4 and the embryonic driver Srp(hemo)Gal4 respectively combined with a lineage tracing construct. The relative expression levels are indicated by the size of the dots and the pvalue of the log2 fold change with the gradient dark blue (not significant) to light blue (p<10-3). The p-value are estimated by bilateral student test and indicated as follow: ns = not-significant (>0.05), "*" = p[0.05 - 0.01[, "**" = p[0.01-0.001[, "***" = p <0.001].


B Western blots of whole - cell extracts showing efficient degradation of PDS5A or PDS5B ( left , right ) with dTAG treatment ( at different time points ) .

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B Western blots of whole-cell extracts showing efficient degradation of PDS5A or PDS5B (left, right) with dTAG treatment (at different time points).


D ChIP - seq signal for FLAG - PDS5A and FLAG - PDSB ( ChIP assays were performed with antibodies against Flag and PDS5A / B ) centered at TSS of promoters ( ± 3 kb ) across all peaks called for each of the proteins in DMSO treated PDS5A - dTAG or PDS5B - dTAG cells , showing the drastic reduction in PDS5A / B enrichment on chromatin after degradation . Top : normalized signal intensities for each protein .

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

D ChIP-seq signal for FLAG-PDS5A and FLAG-PDSB (ChIP assays were performed with antibodies against Flag and PDS5A/B) centered at TSS of promoters (± 3 kb) across all peaks called for each of the proteins in DMSO treated PDS5A-dTAG or PDS5B-dTAG cells, showing the drastic reduction in PDS5A/B enrichment on chromatin after degradation. Top: normalized signal intensities for each protein.


F ChIP - qPCR using Flag antibody demonstrating complete depletion of PDS5A or PDS5B ( left , right ) in H19 / IGF2 imprinting locus and the promoter region of PCDH10 , NC represents a negative binding site . Data are represented as mean ± SD from three biological replicates .

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F ChIP-qPCR using Flag antibody demonstrating complete depletion of PDS5A or PDS5B (left, right) in H19/IGF2 imprinting locus and the promoter region of PCDH10, NC represents a negative binding site. Data are represented as mean±SD from three biological replicates.


B Heatmaps of signal intensities for FLAG - PDS5A , FLAG - PDS5B , CTCF , and RAD21 ChIP - seq data across all defined FLAG - PDS5A peaks .

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B Heatmaps of signal intensities for FLAG-PDS5A, FLAG-PDS5B, CTCF, and RAD21 ChIP-seq data across all defined FLAG-PDS5A peaks.


C Averaged ChIP - seq signal densities of FLAG - PDS5A , FLAG - PDS5B , RAD21 , and CTCF on PDS5A & PDS5B common sites and PDS5A / PDS5B unique sites .

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C Averaged ChIP-seq signal densities of FLAG-PDS5A, FLAG-PDS5B, RAD21, and CTCF on PDS5A & PDS5B common sites and PDS5A/PDS5B unique sites.


F Reciprocal Re - ChIP for PDS5A and PDS5B . The co - binding of PDS5A and PDS5B at PDS5A & PDS5B common sites were assayed by Re - ChIP - qPCR . The second ChIP for Re - ChIP was performed using antibodies against PDS5A , FLAG ( PDS5B ) , RAD21 , control IgG . RAD21 served as a positive control . Data are shown as fold enrichment versus IgG ( mean ± SD from three biological replicates ) .

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F Reciprocal Re-ChIP for PDS5A and PDS5B. The co-binding of PDS5A and PDS5B at PDS5A&PDS5B common sites were assayed by Re-ChIP-qPCR. The second ChIP for Re-ChIP was performed using antibodies against PDS5A, FLAG(PDS5B), RAD21, control IgG. RAD21 served as a positive control. Data are shown as fold enrichment versus IgG (mean±SD from three biological replicates).


A Validation of PDS5A ; PDS5B - dTAG cells for simultaneous depletion of PDS5A and PDS5B .

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A Validation of PDS5A;PDS5B-dTAG cells for simultaneous depletion of PDS5A and PDS5B.


B Chromatin fractionation assay showing the change of chromatin - bound level of RAD21 or CTCF after co - depletion of PDS5A and PDS5B .

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B Chromatin fractionation assay showing the change of chromatin-bound level of RAD21 or CTCF after co-depletion of PDS5A and PDS5B.


C Heatmaps displaying the ChIP - seq binding intensities for significantly changed RAD21 peaks after joint PDS5 proteins depletion ( left ) . Veen diagram showing RAD21 peaks identified in cells before and after co - depletion of PDS5A and PDS5B .

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C Heatmaps displaying the ChIP-seq binding intensities for significantly changed RAD21 peaks after joint PDS5 proteins depletion (left). Veen diagram showing RAD21 peaks identified in cells before and after co-depletion of PDS5A and PDS5B.


D Changes of RAD21 signals at all loop anchors defined in PDS5A - dTAG cells or PDS5B - dTAG cells with DMSO treatment after PDS5A and PDS5B co - depletion , or either depletion . For PDS5A ; PDS5B - dTAG cells , we used the genomic positions of loop anchors identified in DMSO treated PDS5A - dTAG cells . E Differential RAD21 ChIP - seq signals of representative regions showing significantly down - regulated peaks after co - depletion of PDS5A and PDS5B . F Differential RAD21 ChIP - seq signals of representative regions showing significantly up - regulated peaks after co - depletion of PDS5A and PDS5B .

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D Changes of RAD21 signals at all loop anchors defined in PDS5A-dTAG cells or PDS5B-dTAG cells with DMSO treatment after PDS5A and PDS5B co-depletion, or either depletion. For PDS5A;PDS5B-dTAG cells, we used the genomic positions of loop anchors identified in DMSO treated PDS5A-dTAG cells. E Differential RAD21 ChIP-seq signals of representative regions showing significantly down-regulated peaks after co-depletion of PDS5A and PDS5B. F Differential RAD21 ChIP-seq signals of representative regions showing significantly up-regulated peaks after co-depletion of PDS5A and PDS5B.


A , B Volcano plots showing gene expression changes after PDS5A ( A ) and PDS5B ( B ) degradation . Genes with significant changes ( padj < 0 . 05 ) are colored blue ( upregulated ) or red ( downregulated ) .

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A, B Volcano plots showing gene expression changes after PDS5A (A) and PDS5B (B) degradation. Genes with significant changes (padj < 0.05) are colored blue (upregulated) or red (downregulated).


C The overlap and discrepancy of PDS5A and PDS5B misregulated genes ( left ) . Heatmap displaying Benjamini - Hochberg adjusted p - value of shared genes down - regulated and up - regulated after PDS5A or PDS5B depletion ( right ) .

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C The overlap and discrepancy of PDS5A and PDS5B misregulated genes (left). Heatmap displaying Benjamini-Hochberg adjusted p-value of shared genes down-regulated and up-regulated after PDS5A or PDS5B depletion (right).


( A ) HeLa cells were infected with S . Typhimurium ( Salmonella ) for 1 h or transfected with Effectene - coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and transferrin receptor ( TfR ) . Bar , 10 µm .

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(A) HeLa cells were infected with S. Typhimurium (Salmonella) for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and transferrin receptor (TfR). Bar, 10 µm.


( B ) HeLa cells were infected with Salmonella for 1 h or transfected with Effectene - coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and galectin3 . Bar , 5 µm .

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(B) HeLa cells were infected with Salmonella for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and galectin3. Bar, 5 µm.


( C ) HeLa cells were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub ( top ) or LC3 and p62 ( bottom ) . Bar , 5 µm . The percentages of LC3 - or p62 - positive beads per Ub - positive ( Ub + ) or Ub - negative ( Ub − ) beads were enumerated . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 01 .

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(C) HeLa cells were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub (top) or LC3 and p62 (bottom). Bar, 5 µm. The percentages of LC3- or p62-positive beads per Ub-positive (Ub+) or Ub-negative (Ub−) beads were enumerated. Statistical analysis was performed by Student's unpaired t test. *, P < 0.01.


( D and E ) HeLa cells stably expressing GFP - LC3 were transfected with Effectene - coated latex beads for 3 h . Bead - autophagosomes were fractionated as described in Materials and methods . The bead - autophagosome fraction was observed by confocal microscopy ( D ; bar , 10 µm ) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies ( E ) . In a control sample , scraped cells were mixed with Effectene - coated beads and immediately homogenized .

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(D and E) HeLa cells stably expressing GFP-LC3 were transfected with Effectene-coated latex beads for 3 h. Bead-autophagosomes were fractionated as described in Materials and methods. The bead-autophagosome fraction was observed by confocal microscopy (D; bar, 10 µm) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies (E). In a control sample, scraped cells were mixed with Effectene-coated beads and immediately homogenized.


( F ) The bead - autophagosome fraction was lysed and subjected to immunoprecipitation with an anti - Ub IgG antibody ( FK2 ) or control IgG . Co - immunoprecipitated molecules were examined by Western blotting using the indicated antibodies .

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(F) The bead-autophagosome fraction was lysed and subjected to immunoprecipitation with an anti-Ub IgG antibody (FK2) or control IgG. Co-immunoprecipitated molecules were examined by Western blotting using the indicated antibodies.


( G and H ) NIH3T3 cells stably expressing mStrawberry ( mStr ) - Gal3 and GFP - LC3 , mStr - Gal3 , and GFP - p62 , or mStr - Gal3 and GFP - Ub were transfected with Effectene - coated latex beads for 30 min and then washed . Live cells were observed at 1 - min intervals by fluorescence microscopy . Bar , 3 µm . The time after galectin3 localization was measured for at least 30 cases for each combination ( H ) . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 ; NS , not significant .

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(G and H) NIH3T3 cells stably expressing mStrawberry (mStr)-Gal3 and GFP-LC3, mStr-Gal3, and GFP-p62, or mStr-Gal3 and GFP-Ub were transfected with Effectene-coated latex beads for 30 min and then washed. Live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after galectin3 localization was measured for at least 30 cases for each combination (H). Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.


( A and B ) NIH3T3 cells stably expressing mStr - Ub and GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , or ULK1 were transfected with Effectene - coated latex beads for 30 min . Then , live cells were observed at 1 - min intervals by fluorescence microscopy . Bar , 3 µm . The time after Ub localization was measured for at least 30 cases for each combination . Each value represents the mean ± SD . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 ; NS , not significant .

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(A and B) NIH3T3 cells stably expressing mStr-Ub and GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, or ULK1 were transfected with Effectene-coated latex beads for 30 min. Then, live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after Ub localization was measured for at least 30 cases for each combination. Each value represents the mean ± SD. Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.


( C ) NIH3T3 cells stably expressing GFP - tagged Ub , LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated beads for 3 h in the presence or absence ( mock ) of 30 µM UBEI - 41 ( a ubiquitin E1 - specific inhibitor ) and subjected to immunocytochemistry for galectin3 . The percentages of GFP - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 .

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(C) NIH3T3 cells stably expressing GFP-tagged Ub, LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated beads for 3 h in the presence or absence (mock) of 30 µM UBEI-41 (a ubiquitin E1-specific inhibitor) and subjected to immunocytochemistry for galectin3. The percentages of GFP-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD. Statistical analysis was performed by Student's unpaired t test. *, P < 0.05.


( D and E ) Parent NIH3T3 cells , Atg4B mutant overexpressing NIH3T3 cells stably expressing GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for galectin3 . The percentages of Atg - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD .

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(D and E) Parent NIH3T3 cells, Atg4B mutant overexpressing NIH3T3 cells stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.


( E ) wild - type MEFs , and Atg5 - KO MEFs stably expressing GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for galectin3 . The percentages of Atg - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD .

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(E) wild-type MEFs, and Atg5-KO MEFs stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.


( A ) HeLa cells were lysed with 1 % Triton X - 100 lysis buffer and subjected to immunoprecipitation with FK2 - or control IgG - immobilized beads . The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies .

(A) HeLa cells were lysed with 1% Triton X-100 lysis buffer and subjected to immunoprecipitation with FK2- or control IgG-immobilized beads. The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies.


( C ) Purified recombinant GST or GST - Ub - immobilized glutathione Sepharose beads were incubated with lysates from HEK293T cells transiently expressing FLAG - tagged Atg16L1 constructs for 1 h at 25 ° C with gentle agitation . The beads were washed three times with ice - cold PBS and the bound complexes were eluted with 50 mM reduced glutathione and then subjected to Western blotting for FLAG .

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(C) Purified recombinant GST or GST-Ub-immobilized glutathione Sepharose beads were incubated with lysates from HEK293T cells transiently expressing FLAG-tagged Atg16L1 constructs for 1 h at 25°C with gentle agitation. The beads were washed three times with ice-cold PBS and the bound complexes were eluted with 50 mM reduced glutathione and then subjected to Western blotting for FLAG.


( D ) Purified GST or GST - Ub - immobilized glutathione Sepharose beads were incubated with lysates from bacteria expressing trigger factor ( TF ) or TF - FLAG - WD β - propellers for 1 h at 25 ° C with gentle agitation . Washing and elution were performed as described in C . The eluted samples were subjected to SDS - PAGE and Western blotting for FLAG .

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(D) Purified GST or GST-Ub-immobilized glutathione Sepharose beads were incubated with lysates from bacteria expressing trigger factor (TF) or TF-FLAG-WD β-propellers for 1 h at 25°C with gentle agitation. Washing and elution were performed as described in C. The eluted samples were subjected to SDS-PAGE and Western blotting for FLAG.


( A ) Yeast two - hybrid interactions of the ULK1 complexes with other human Atg proteins . ULK1 , ULK2 , Atg13 , FIP200 , and Atg101 - AD fusions ( rows 2 - 6 ) or control AD constructs ( row 1 , Empty ) were coexpressed with control DBD constructs ( column 1 , Empty ) or Atg protein - DBD fusions ( columns 2 - 35 ) and tested for positive yeast two - hybrid interactions ( top ) or cotransformation ( control , bottom ) . White lines divide images derived from the same plate , and red lines divide images from different plates . Atg16L1 positively interacted with FIP200 in this assay .

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(A) Yeast two-hybrid interactions of the ULK1 complexes with other human Atg proteins. ULK1, ULK2, Atg13, FIP200, and Atg101-AD fusions (rows 2-6) or control AD constructs (row 1, Empty) were coexpressed with control DBD constructs (column 1, Empty) or Atg protein-DBD fusions (columns 2-35) and tested for positive yeast two-hybrid interactions (top) or cotransformation (control, bottom). White lines divide images derived from the same plate, and red lines divide images from different plates. Atg16L1 positively interacted with FIP200 in this assay.


( B ) FIP200 binds to exogenously expressed Atg16L1 . Myc - Atg16L1 coprecipitations with an empty vector control ( lane 1 ) or One - STrEP - FLAG ( OSF ) - FIP200 ( lane 2 ) .

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(B) FIP200 binds to exogenously expressed Atg16L1. Myc-Atg16L1 coprecipitations with an empty vector control (lane 1) or One-STrEP-FLAG (OSF)-FIP200 (lane 2).


( C ) FIP200 binds endogenous Atg16L1 . Atg16L1 coprecipitations with an empty vector control ( lane 1 ) or OSF - FIP200 ( lane 2 ) .

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(C) FIP200 binds endogenous Atg16L1. Atg16L1 coprecipitations with an empty vector control (lane 1) or OSF-FIP200 (lane 2).


( D ) Wild - type or FIP200 - KO MEFs stably expressing the empty vector , full - length Atg16L1 , or ΔWD mutant were incubated in growth medium ( − ) or Earle ' s balanced salt solution ( EBSS ) ( + ) for 1 h and examined by Western blotting using the indicated antibodies . We previously reported that stable expression of an exogenous Atg16L1 construct can be used to replace the endogenous Atg16L1 protein with an exogenous Atg16L1 protein ( Fujita et al . , 2009 ) because free Atg16L1 molecules not complexed with the Atg12 - Atg5 conjugate are preferentially degraded by the ubiquitin - proteasome system . In control cells ( Empty ) , two Atg16L1 splicing variants , the α - and β - forms , were detected . In full - length β - form - replaced cells ( Full ) , the α - form was not detected , whereas in ΔWD - replaced cells ( ΔWD ) both the α - and β - forms were minimally detected . Thus , we successfully obtained cells that express only endogenous levels of the full - length or ΔWD mutant .

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(D) Wild-type or FIP200-KO MEFs stably expressing the empty vector, full-length Atg16L1, or ΔWD mutant were incubated in growth medium (−) or Earle's balanced salt solution (EBSS) (+) for 1 h and examined by Western blotting using the indicated antibodies. We previously reported that stable expression of an exogenous Atg16L1 construct can be used to replace the endogenous Atg16L1 protein with an exogenous Atg16L1 protein (Fujita et al., 2009) because free Atg16L1 molecules not complexed with the Atg12-Atg5 conjugate are preferentially degraded by the ubiquitin-proteasome system. In control cells (Empty), two Atg16L1 splicing variants, the α- and β-forms, were detected. In full-length β-form-replaced cells (Full), the α-form was not detected, whereas in ΔWD-replaced cells (ΔWD) both the α- and β-forms were minimally detected. Thus, we successfully obtained cells that express only endogenous levels of the full-length or ΔWD mutant.


( E - L ) Atg16L1 - replaced wild - type or FIP200 - KO MEFs were infected with Salmonella ( MOI = 10 ) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub ( E and F ) or LC3 and Ub ( I and J ) . The percentages of Atg16L1 - positive ( G and H ) or LC3 - positive per Ub - positive bacteria were enumerated ( K and L ) . At least 50 Salmonella were counted . The average ± SD is shown for three independent experiments . Statistical analysis was performed by Student ' s t test . * , P < 0 . 05 ; NS , not significant . Bar , 5 µm .

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(E-L) Atg16L1-replaced wild-type or FIP200-KO MEFs were infected with Salmonella (MOI = 10) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub (E and F) or LC3 and Ub (I and J). The percentages of Atg16L1-positive (G and H) or LC3-positive per Ub-positive bacteria were enumerated (K and L). At least 50 Salmonella were counted. The average ± SD is shown for three independent experiments. Statistical analysis was performed by Student's t test. *, P < 0.05; NS, not significant. Bar, 5 µm.


( A ) Mapping of the Atg16L1 - binding site on FIP200 . Yeast two - hybrid mapping experiments showing that Atg16L1 binds to both the N - terminal and C - terminal coiled - coil regions of FIP200 .

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(A) Mapping of the Atg16L1-binding site on FIP200. Yeast two-hybrid mapping experiments showing that Atg16L1 binds to both the N-terminal and C-terminal coiled-coil regions of FIP200.


( B ) Mapping of the FIP200 - binding site within Atg16L1 . Yeast two - hybrid mapping experiments showing that the 1 - 840 fragment of FIP200 binds to the C - terminal 247 - 607 region , while the 1276 - 1591 fragment of FIP200 binds to the N - terminal 1 - 264 region of Atg16L1 .

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(B) Mapping of the FIP200-binding site within Atg16L1. Yeast two-hybrid mapping experiments showing that the 1-840 fragment of FIP200 binds to the C-terminal 247-607 region, while the 1276-1591 fragment of FIP200 binds to the N-terminal 1-264 region of Atg16L1.


( C and D ) Additional yeast two - hybrid mapping experiments showing that the binding site of the FIP200 1276 - 1591 fragment maps to residues 239 - 246 of Atg16L1 . White lines divide images derived from the same plate , and red lines divide images from different plates .

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(C and D) Additional yeast two-hybrid mapping experiments showing that the binding site of the FIP200 1276-1591 fragment maps to residues 239-246 of Atg16L1. White lines divide images derived from the same plate, and red lines divide images from different plates.


( E ) The N - terminal 1 - 249 fragment of Atg16L1 is sufficient to interact with FIP200 . Full - length ( lanes 1 and 2 ) or the N - terminal 1 - 249 fragments ( lanes 3 and 4 ) of Myc - Atg16L1 coprecipitations with empty vector controls ( lanes 1 and 3 ) or OSF - FIP200 1276 - 1591 ( lanes 2 and 4 ) .

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(E) The N-terminal 1-249 fragment of Atg16L1 is sufficient to interact with FIP200. Full-length (lanes 1 and 2) or the N-terminal 1-249 fragments (lanes 3 and 4) of Myc-Atg16L1 coprecipitations with empty vector controls (lanes 1 and 3) or OSF-FIP200 1276-1591 (lanes 2 and 4).


( F ) Co - precipitation experiments confirming that OSF - FIP200 1276 - 1591 coprecipitates with wild - type ( WT ) Myc - Atg16L1 ( 1 - 249 ) fragments , but not with analogous fragments containing a mutant FIP200 - binding site ( Myc - Atg16L1 ΔWD 239 - 242A ) .

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(F) Co-precipitation experiments confirming that OSF-FIP200 1276-1591 coprecipitates with wild-type (WT) Myc-Atg16L1 (1-249) fragments, but not with analogous fragments containing a mutant FIP200-binding site (Myc-Atg16L1 ΔWD 239-242A).


( B - E ) Atg16L1 - Δ / Δ MEFs stably expressing the indicated constructs were infected with Salmonella ( MOI = 10 ) for 1 h and then analyzed by immunocytochemistry for LC3 ( B ) or Atg16L1 ( D ) . Bar , 5 µm . The percentage of LC3 - or Atg16L1 - positive Salmonella per Ub - positive Salmonella was enumerated by fluorescence microscopy ( C and E ) . At least 50 bacteria were counted . The average ± SD is shown for three independent experiments .

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(B-E) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were infected with Salmonella (MOI = 10) for 1 h and then analyzed by immunocytochemistry for LC3 (B) or Atg16L1 (D). Bar, 5 µm. The percentage of LC3- or Atg16L1-positive Salmonella per Ub-positive Salmonella was enumerated by fluorescence microscopy (C and E). At least 50 bacteria were counted. The average ± SD is shown for three independent experiments.


( F ) Atg16L1 - Δ / Δ MEFs stably expressing the indicated constructs were cultured in growth medium ( Fed ) or EBSS ( Starved ) for 1 h and then subjected to immunocytochemistry using an anti - LC3 antibody . The number of LC3 puncta in each cell was counted for more than 50 cells . The average ± SD is shown for three independent experiments .

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(F) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were cultured in growth medium (Fed) or EBSS (Starved) for 1 h and then subjected to immunocytochemistry using an anti-LC3 antibody. The number of LC3 puncta in each cell was counted for more than 50 cells. The average ± SD is shown for three independent experiments.


A ) Representative size - exclusion chromatography ( SEC ) chromatogram with absorbance measured at nanometers ( nm ) 260 or 280 ( for this chromatogram and the following ones , absorbances are in units of 1000 - 1 ( mAU ) ) of reconstituted K . lactis COMA - Nkp1 - Nkp2 , and image of Coomassie Blue - stained SDS - PAGE gel with fractions from principal SEC peaks . Molecular masses of protein standards ( Mm . S . ) are in kiloDalton ( kDa ) ; L : sample loaded on column . † : Escherichia Coli DnaK ; ╪ : proteolysed Nkp1 - Nkp2 ; Nkp1 - Nkp2 is less prone to spontaneous proteolysis when associated with COMA .

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A) Representative size-exclusion chromatography (SEC) chromatogram with absorbance measured at nanometers (nm) 260 or 280 (for this chromatogram and the following ones, absorbances are in units of 1000-1 (mAU)) of reconstituted K. lactis COMA-Nkp1-Nkp2, and image of Coomassie Blue-stained SDS-PAGE gel with fractions from principal SEC peaks. Molecular masses of protein standards (Mm. S.) are in kiloDalton (kDa); L: sample loaded on column. †: Escherichia Coli DnaK; ╪: proteolysed Nkp1-Nkp2; Nkp1-Nkp2 is less prone to spontaneous proteolysis when associated with COMA.


B , C ) Nanoflow mass - spectra of COMA - Nkp1 - Nkp2 ( B ) acquired with different injected concentrations , showing an increase in COMA - Nkp1 - Nkp2 dimers or COMA dimers with increasing injectant concentration .

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B,C) Nanoflow mass-spectra of COMA-Nkp1-Nkp2 (B) acquired with different injected concentrations, showing an increase in COMA-Nkp1-Nkp2 dimers or COMA dimers with increasing injectant concentration.


B , C ) Nanoflow mass - spectra of COMA ( C ) , acquired with different injected concentrations , showing an increase in COMA - Nkp1 - Nkp2 dimers or COMA dimers with increasing injectant concentration .

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B,C) Nanoflow mass-spectra of COMA (C), acquired with different injected concentrations, showing an increase in COMA-Nkp1-Nkp2 dimers or COMA dimers with increasing injectant concentration.


D ) Graph with measured differential refractive index ( relative scale ) and molar mass calculated with multi - angle light scattering ( MALS ) data of COMA eluting from SEC ( SEC - MALS ) . We injected COMA at a concentration of ~ 43 μM ( COMA dimer ) on the column . Dashed lines indicate expected molar masses for monomeric COMA or dimeric COMA : 142337 g mol − 1 or 284674 g mol − 1 ; our experimentally determined molar mass from the principal SEC elution peak : 299100 g mol − 1 ± 5683 g mol − 1 ( mean ± standard deviation from a single SEC - MALS experiment ) ; max . dimeric COMA concentration , from refractive index measurement , in principal peak : 2 . 4 μM .

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D) Graph with measured differential refractive index (relative scale) and molar mass calculated with multi-angle light scattering (MALS) data of COMA eluting from SEC (SEC-MALS). We injected COMA at a concentration of ~ 43 μM (COMA dimer) on the column. Dashed lines indicate expected molar masses for monomeric COMA or dimeric COMA: 142337 g mol−1 or 284674 g mol−1; our experimentally determined molar mass from the principal SEC elution peak: 299100 g mol−1 ± 5683 g mol−1 (mean ± standard deviation from a single SEC-MALS experiment); max. dimeric COMA concentration, from refractive index measurement, in principal peak: 2.4 μM.


A ) Excerpt from a representative nanoflow mass - spectrum of COMA ( full spectrum and enlarged spectrum areas are in Appendix Fig S2A ) ; B ) mass - spectrum from tandem mass - spectrometry of the isolated 28 + charge state of COMA ( from spectrum shown in A ) , after its collision - induced dissociation in the gas phase . Charge states of assigned masses are indicated above peaks . For this spectrum and our other spectra of this type , charge states of assigned masses are indicated above peaks . Inset table shows identified proteins or protein assemblies , their measured mass values as mean ± standard deviation from multiple peaks in a peak series assigned to the same molecular species ; and their respective expected mass .

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A) Excerpt from a representative nanoflow mass-spectrum of COMA (full spectrum and enlarged spectrum areas are in Appendix Fig S2A); B) mass-spectrum from tandem mass-spectrometry of the isolated 28+ charge state of COMA (from spectrum shown in A), after its collision-induced dissociation in the gas phase. Charge states of assigned masses are indicated above peaks. For this spectrum and our other spectra of this type, charge states of assigned masses are indicated above peaks. Inset table shows identified proteins or protein assemblies, their measured mass values as mean ± standard deviation from multiple peaks in a peak series assigned to the same molecular species; and their respective expected mass.


A , B ) Plots showing hydrogen - deuterium exchanged peptides , after 10 sec of deuterium exchange of Okp1 ( A ) each in COMA or COMA - Nkp1 - Nkp2 . For this representation , and all following of this type , detected peptides are colour - coded according to their measured deuterium - exchange in % of the maximum measured exchange ( 1 - 25 % : dark blue ; 26 - 50 % : light blue ; 51 - 75 % : yellow ; 76 - 100 % : red ) . Peptides are represented as bars . Each bar is plotted row - wise corresponding to its position in the amino - acid sequence of the protein it derives from . Amino - acid sequence scheme is illustrated at the top with thin or thick lines ; thin line : flexible element : thick line : structured segment . Positions of predicted coiled coils are shown as helices . Plots for full time courses are in Appendix Fig S3C , E .

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A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Okp1 (A) each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E.


A , B ) Plots showing hydrogen - deuterium exchanged peptides , after 10 sec of deuterium exchange of Ame1 ( B ) , each in COMA or COMA - Nkp1 - Nkp2 . For this representation , and all following of this type , detected peptides are colour - coded according to their measured deuterium - exchange in % of the maximum measured exchange ( 1 - 25 % : dark blue ; 26 - 50 % : light blue ; 51 - 75 % : yellow ; 76 - 100 % : red ) . Peptides are represented as bars . Each bar is plotted row - wise corresponding to its position in the amino - acid sequence of the protein it derives from . Amino - acid sequence scheme is illustrated at the top with thin or thick lines ; thin line : flexible element : thick line : structured segment . Positions of predicted coiled coils are shown as helices . Plots for full time courses are in Appendix Fig S3C , E .

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A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Ame1 (B), each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E.


B , C ) Representative images of Coomassie Blue - stained SDS - PAGE gels with Ni2 + - affinity purification eluates . B ) of full - length COMA ( lane 11 ) with full - length polyhistidine - tagged Mcm21 ( His - Mcm21 ) and Ctf19 , or COMA variants with polyhistidine - tagged Mcm21 D - RWD domain ( His - Mcm21 _ D - RWD ; K . lactis Mcm21 residues 108 - 293 ) and Ctf19 D - RWD domain ( Ctf19 _ D - RWD ; K . lactis Ctf19 residues 107 - 270 ) We show one - letter identifiers for protein versions in A . Lanes between lanes 5 and 6 are not relevant .

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B,C) Representative images of Coomassie Blue-stained SDS-PAGE gels with Ni2+-affinity purification eluates. B) of full-length COMA (lane 11) with full-length polyhistidine-tagged Mcm21 (His-Mcm21) and Ctf19, or COMA variants with polyhistidine-tagged Mcm21 D-RWD domain (His-Mcm21_D-RWD; K. lactis Mcm21 residues 108-293) and Ctf19 D-RWD domain (Ctf19_D-RWD; K. lactis Ctf19 residues 107-270) We show one-letter identifiers for protein versions in A. Lanes between lanes 5 and 6 are not relevant.


B , C ) Representative images of Coomassie Blue - stained SDS - PAGE gels with Ni2 + - affinity purification eluates . C ) of Ame1 - Okp1 variants with polyhistidine - tagged Okp1 variants ( His - Okp1 variants ) . We show one - letter identifiers for protein versions in A . Lanes between lanes 5 and 6 are not relevant .

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B,C) Representative images of Coomassie Blue-stained SDS-PAGE gels with Ni2+-affinity purification eluates. C) of Ame1-Okp1 variants with polyhistidine-tagged Okp1 variants (His-Okp1 variants). We show one-letter identifiers for protein versions in A. Lanes between lanes 5 and 6 are not relevant.


D ) Image of SDS - PAGE gel with fractions of two purified Ctf19 - Mcm21 - Okp1 variants ( after SEC ) ; expected migrating position of non - proteolysed Okp1 variant ( compare with its position in SDS - PAGE images of COMA or Ame1 - Okp1 variants in B or C ) is marked ; 2 and 3 : SEC - purified fractions of samples from different elution fractions from ion - exchange chromatography . When we co - expressed full - length Okp1 with Ctf19 and Mcm21 , which has a coding regions for an N - terminal polyhistidine - tag , we did not co - purify substantial amounts of full - length Okp1 with Ctf19 - Mcm21 , probably because Okp1 was prone to proteolysis ( in the absence of Ame1 ) .

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D) Image of SDS-PAGE gel with fractions of two purified Ctf19-Mcm21-Okp1 variants (after SEC); expected migrating position of non-proteolysed Okp1 variant (compare with its position in SDS-PAGE images of COMA or Ame1-Okp1 variants in B or C) is marked; 2 and 3: SEC-purified fractions of samples from different elution fractions from ion-exchange chromatography. When we co-expressed full-length Okp1 with Ctf19 and Mcm21, which has a coding regions for an N-terminal polyhistidine-tag, we did not co-purify substantial amounts of full-length Okp1 with Ctf19-Mcm21, probably because Okp1 was prone to proteolysis (in the absence of Ame1).


A , B ) Overlayed chromatograms showing absorbance at 280 nm from SEC of Ctf19 - Mcm21 - Okp1 variants without Okp1 segment 3 ( A ) or with Okp1 segment 3 ( B ) ; Ctf19 - Mcm21 - Okp1 variants : CMO , Nkp1 - Nkp2 : NN , Ctf19 - Mcm21 - Okp1 variants combined with Nkp1 - Nkp2 : CMO - NN ; and images of SDS - PAGE gels with equivalent elution fractions from five SEC experiments ; L : loaded on column . The elution - volume range that we analysed fractions of on SDS - PAGE is indicated below the chromatograms .

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A,B) Overlayed chromatograms showing absorbance at 280 nm from SEC of Ctf19-Mcm21-Okp1 variants without Okp1 segment 3 (A) or with Okp1 segment 3 (B); Ctf19-Mcm21-Okp1 variants: CMO, Nkp1-Nkp2: NN, Ctf19-Mcm21-Okp1 variants combined with Nkp1-Nkp2: CMO-NN; and images of SDS-PAGE gels with equivalent elution fractions from five SEC experiments; L: loaded on column. The elution-volume range that we analysed fractions of on SDS-PAGE is indicated below the chromatograms.


C ) Representative microscopy - images of living haploid S . cerevisiae cells with Okp1 _ fl ( integrated full - length Okp1 ) or our Okp1 version that lacks the coding region for segment 3 - Okp1 _ nnΔ , with Nkp1 - GFP or Nkp2 - GFP . Fluorescence images are maximum intensity projected , and we show those from Okp1 _ fl or Okp1 _ nnΔ with the same fluorophor - fusion kinetochore protein on the same intensity scale ; scale bar : 2 μm .

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C) Representative microscopy-images of living haploid S. cerevisiae cells with Okp1_fl (integrated full-length Okp1) or our Okp1 version that lacks the coding region for segment 3-Okp1_nnΔ, with Nkp1-GFP or Nkp2-GFP. Fluorescence images are maximum intensity projected, and we show those from Okp1_fl or Okp1_nnΔ with the same fluorophor-fusion kinetochore protein on the same intensity scale; scale bar: 2 μm.