B , C , Mouse endothelial cells ( mHEVc ; B ) and human retinal pigment epithelial cells ( RPE , C ) were challenged with RH T . gondii followed by assessment of Akt phosphorylation .

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B, C, Mouse endothelial cells (mHEVc; B) and human retinal pigment epithelial cells (RPE, C) were challenged with RH T. gondii followed by assessment of Akt phosphorylation.


D , HBMEC were challenged with either live or killed T . gondii . Cell lysates were subject to immunoblotting as indicated .

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D, HBMEC were challenged with either live or killed T. gondii. Cell lysates were subject to immunoblotting as indicated.


E , HBMEC were transiently transfected with a plasmid encoding PH - Akt - GFP . Cells treated with or without LY294002 ( 20 µM ) for 1 h prior to challenge with T . gondii - RFP . Cells were examined by immunofluorescence at 5 min post - challenge to examine accumulation of PH - Akt - GFP around the parasites .

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E, HBMEC were transiently transfected with a plasmid encoding PH-Akt-GFP. Cells treated with or without LY294002 (20 µM) for 1 h prior to challenge with T. gondii-RFP. Cells were examined by immunofluorescence at 5 min post-challenge to examine accumulation of PH-Akt-GFP around the parasites.


F , HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T . gondii . Densitometry data represent means ± SEM of 4 experiments . A vertical line was inserted between densitometry data from control and LY294002 - treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 .

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F, HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T. gondii. Densitometry data represent means ± SEM of 4 experiments. A vertical line was inserted between densitometry data from control and LY294002-treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1.


G , HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA . Cells expressing either control siRNA or PI3K p110α siRNA were challenged with T . gondii 48 h after transfection . Cell lysates were subject to immunoblotting as indicated . Densitometry data are shown as above and represent means ± SEM of 3 experiments . Results shown are representative of 3 - 4 independent experiments .

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G, HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA. Cells expressing either control siRNA or PI3K p110α siRNA were challenged with T. gondii 48 h after transfection. Cell lysates were subject to immunoblotting as indicated. Densitometry data are shown as above and represent means ± SEM of 3 experiments. Results shown are representative of 3-4 independent experiments.


A , HBMEC , mHEVc and human RPE cells were incubated with or without Akt inhibitor IV ( 1 . 25 µM ) for 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy 2 h and 24 h post - challenge .

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A, HBMEC, mHEVc and human RPE cells were incubated with or without Akt inhibitor IV (1.25 µM) for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.


B , HBMEC were transfected with control siRNA or Akt siRNA . Cells were then challenged with T . gondii 48 h after transfection . Monolayers were examined microscopically 24 h post - challenge .

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B, HBMEC were transfected with control siRNA or Akt siRNA. Cells were then challenged with T. gondii 48 h after transfection. Monolayers were examined microscopically 24 h post-challenge.


C , RAW 264 . 7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy 2 h and 24 h post - challenge .

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C, RAW 264.7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.


D , mHEVc - LC3 - EGFP cells were incubated with or without Akt inhibitor IVfollowed by challenge with T . gondii - RFP . Monolayers were examined by fluorescence microscopy 5 h post - challenge . Arrowheads indicate accumulation of LC3 around the parasite .

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D, mHEVc-LC3-EGFP cells were incubated with or without Akt inhibitor IVfollowed by challenge with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite.


E , HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii ( T ) and then processed for electron microscopy at 5 h post - challenge . Images at the bottom represent magnification of the areas within the boxes . Arrow indicates the PVM ; arrowhead indicates the double membrane structure around the vacuole .

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E, HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole.


F , Control or Akt inhibitor IV - treated HBMEC were challenged with T . gondii - YFP . Expression of LAMP - 1 was examined by fluorescent microscopy 8 h post - challenge . Arrowheads indicate accumulation of LAMP - 1 around the

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F, Control or Akt inhibitor IV-treated HBMEC were challenged with T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasite


G , H , mHEVc cells were transfected with Beclin 1 siRNA ( G ) , Atg7 siRNA ( H ) or control siRNA . After 48 h , cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy at 24 h .

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G, H, mHEVc cells were transfected with Beclin 1 siRNA (G), Atg7 siRNA (H) or control siRNA. After 48 h, cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 24 h.


I , mHEVc were treated with or without Akt inhibitor IV and infected with T . gondii . 1 h post infection cells were treated with or without leupeptin plus pepstatin ( Lys inhibitors ) . Monolayers were examined microscopically 24 h post - challenge .

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I, mHEVc were treated with or without Akt inhibitor IV and infected with T. gondii. 1 h post infection cells were treated with or without leupeptin plus pepstatin (Lys inhibitors). Monolayers were examined microscopically 24 h post-challenge.


J , Mouse microglia were incubated with or without Akt inhibitor IV . 3 - methyl adenine ( 3MA ; 10 mM ) or vehicle were added 2 h post - challenge with T . gondii . Monolayers were examined microscopically 24 h post - challenge . Results are shown as the mean ± SEM and are representative of 3 independent experiments .

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J, Mouse microglia were incubated with or without Akt inhibitor IV. 3-methyl adenine (3MA; 10 mM) or vehicle were added 2 h post-challenge with T. gondii. Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.


A , HBMEC were treated with or without pertussis toxin ( PTx ; 100 ng / mL ) for 4 h prior to challenge with T . gondii . Cell lysates were obtained at 5 , 15 or 30 min post - incubation with T . gondii and used to examine total Akt and phospho - Akt serine 473 by immunoblot . Densitometry data represent means ± SEM of 3 experiments . A vertical line was inserted between densitometry data from control and PTx - treated cells to indicate that band densities from infected cells treated with or without PTx were compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . HBMEC were also treated with or without LPA ( 10 µM ) in the presence or absence of PTx . Cell lysates were obtained at 5 min and subjected to

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A, HBMEC were treated with or without pertussis toxin (PTx; 100 ng/mL) for 4 h prior to challenge with T. gondii. Cell lysates were obtained at 5, 15 or 30 min post-incubation with T. gondii and used to examine total Akt and phospho-Akt serine 473 by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and PTx-treated cells to indicate that band densities from infected cells treated with or without PTx were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. HBMEC were also treated with or without LPA (10 µM) in the presence or absence of PTx. Cell lysates were obtained at 5 min and subjected to immunoblotting


B , HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h . Cell lysates were used to examine total Akt and phospho - serine 473 Akt by immunoblot . Results shown are representative of 3 independent experiments .

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B, HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h. Cell lysates were used to examine total Akt and phospho-serine 473 Akt by immunoblot. Results shown are representative of 3 independent experiments.


A , HBMEC cells were challenged with RH T . gondii and cell lysates were obtained to probe for total EGFR , phospho - tyrosine 1068 EGFR and phospho - tyrosine 1148 EGFR by immunoblot .

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A, HBMEC cells were challenged with RH T. gondii and cell lysates were obtained to probe for total EGFR, phospho-tyrosine 1068 EGFR and phospho- tyrosine 1148 EGFR by immunoblot.


B , HBMEC were challenged with live vs killed tachyzoites . Total EGFR and phospho - tyrosine 1068 EGFR expression was examined by immunoblot .

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B, HBMEC were challenged with live vs killed tachyzoites. Total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot.


C , D , Human RPE cells ( C ) and mouse microglia ( D ) were challenged with RH T . gondii and total EGFR and phospho - tyrosine 1068 EGFR expression was examined by immunoblot . Results shown are representative of 3 independent experiments .

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C, D, Human RPE cells (C) and mouse microglia (D) were challenged with RH T. gondii and total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot. Results shown are representative of 3 independent experiments.


A , mHEVc cells were transfected with control siRNA or EGFR siRNA . Expression of EGFR and actin were assessed by immunoblot 48 h post - transfection . mHEVc cells expressing either control siRNA or EGFR siRNA were challenged with T . gondii . Cell lysates were used to examine the expression total Akt or phospho - Ser473 Akt by immunoblot .

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A, mHEVc cells were transfected with control siRNA or EGFR siRNA. Expression of EGFR and actin were assessed by immunoblot 48 h post-transfection. mHEVc cells expressing either control siRNA or EGFR siRNA were challenged with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot.


B , Mouse microglia were treated with or without AG1478 ( 1 µM ) 1 h prior to challenge with T . gondii . Cell lysates were used to examine the expression total Akt or phospho - Ser473 Akt by immunoblot . Densitometry data represent means ± SEM of 3 experiments . A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478 - treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . Results shown are representative of 3 independent experiments .

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B, Mouse microglia were treated with or without AG1478 (1 µM) 1 h prior to challenge with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478-treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.


A , HBMEC were incubated with AG1478 ( 1 µM ) 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy at 2 and 24 h .

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A, HBMEC were incubated with AG1478 (1 µM) 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.


B , Human RPE cells transfected with either EGFR or control siRNA were challenged with T . gondii followed by examination of monolayers by light microscopy at 24 h .

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B, Human RPE cells transfected with either EGFR or control siRNA were challenged with T. gondii followed by examination of monolayers by light microscopy at 24 h.


C , Mouse bone marrow - derived macrophages were incubated with AG1478 and challenged with T . gondii . Monolayers were examined by light microscopy at 2 and 24 h .

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C, Mouse bone marrow-derived macrophages were incubated with AG1478 and challenged with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.


D , Parental CHO and CHO - EGFR cells were challenged with RH T . gondii . Monolayers were examined microscopically 2 h or 24 h post - challenge .

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D, Parental CHO and CHO-EGFR cells were challenged with RH T. gondii. Monolayers were examined microscopically 2 h or 24 h post-challenge.


E , mHEVc - LC3 - EGFP cells transfected with control siRNA or EGFR siRNA were challenged with T . gondii - RFP . Monolayers were examined by fluorescence microscopy 5 h post - challenge to determine the percentage of endothelial cells with LC3 accumulation around the parasite .

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E, mHEVc-LC3-EGFP cells transfected with control siRNA or EGFR siRNA were challenged with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge to determine the percentage of endothelial cells with LC3 accumulation around the parasite.


F , HBMEC cells treated with or without AG1478 were challenged with either T . gondii - YFP . Expression of LAMP - 1 was examined by fluorescent microscopy 8 h post - challenge . The percentages of endothelial cells with LAMP - 1 accumulation of around the parasite were determined .

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F, HBMEC cells treated with or without AG1478 were challenged with either T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. The percentages of endothelial cells with LAMP-1 accumulation of around the parasite were determined.


G , H , mHEVc cells transfected with Beclin1 siRNA ( G ) or Atg7 siRNA ( H ) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T . gondii . Monolayers were examined by light microscopy 24 h post - challenge . Results are shown as the mean ± SEM and are representative of 3 independent experiments .

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G, H, mHEVc cells transfected with Beclin1 siRNA (G) or Atg7 siRNA (H) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T. gondii. Monolayers were examined by light microscopy 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.


HBMEC were treated with or without GM6001 ( 10 µM ) for 1 h prior to challenge with T . gondii ( A ) or with Pertussis Toxin ( PTx ; 100 ng / ml ) for 4 h prior to parasite challenge ( B ) . Cell lysates were used to examine total EGFR and phospho - tyrosine 1068 EGFR by immunoblot . Densitometry data represent means ± SEM of 3 experiments . A vertical line was inserted between densitometry data from control and GM6001 - or PTX - treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . Results shown are representative of 3 independent experiments .

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HBMEC were treated with or without GM6001 (10 µM) for 1 h prior to challenge with T. gondii (A) or with Pertussis Toxin (PTx; 100 ng/ml) for 4 h prior to parasite challenge (B). Cell lysates were used to examine total EGFR and phospho-tyrosine 1068 EGFR by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and GM6001- or PTX-treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.


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A , B , C HBMEC were challenged with ΔHx ( WT ) , MIC1 ko , MIC3 ko , MIC1 - 3 ko T . gondii at MOIs that yielded similar percentages of infected cells ( A ) .

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A, B, C HBMEC were challenged with ΔHx (WT), MIC1 ko, MIC3 ko, MIC1-3 ko T. gondii at MOIs that yielded similar percentages of infected cells (A).


Cell lysates were obtained and used to examine total EGFR and phospho - tyrosine 1068 EGFR ( B ) or total Akt and phospho - Ser 473 Akt ( C ) by immunoblot .

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Cell lysates were obtained and used to examine total EGFR and phospho-tyrosine 1068 EGFR (B) or total Akt and phospho-Ser 473 Akt (C) by immunoblot.


HBMEC were incubated with 10 nM of recombinant MIC4 , MIC3 , M2AP or MIC6 for 15 minutes followed by examination of total EGFR and phospho - tyrosine 1068 EGFR ( D ) or total Akt and phospho - Ser473 ( E ) by immunoblot . Densitometry data represent means ± SEM of 4 experiments . Band densities from infected cells or cells treated with MICs were compared to bands from uninfected or untreated cells ( Un ) , which were given an arbitrary number of 1 . Results shown are representative of 4 independent experiments .

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HBMEC were incubated with 10 nM of recombinant MIC4, MIC3, M2AP or MIC6 for 15 minutes followed by examination of total EGFR and phospho-tyrosine 1068 EGFR (D) or total Akt and phospho-Ser473 (E) by immunoblot. Densitometry data represent means ± SEM of 4 experiments. Band densities from infected cells or cells treated with MICs were compared to bands from uninfected or untreated cells (Un), which were given an arbitrary number of 1. Results shown are representative of 4 independent experiments.


A , hmCD40 mHEVc expressing LC3 - EGFP were treated with or without CD154 followed by challenge with T . gondii - RFP in the presence or absence of EGF ( 50 ng / ml ) or AG1478 ( 1 µM ) . LC3 accumulation around T . gondii was assessed by immunofluorescence . B , mHEVc - LC3 - EGFP cells were infected with T . gondii - T . gondii . Cells were treated with vehicle or rapamycin ( 1 µM ) 2 h after challenge with T . gondii in the presence or absent of EGF or AG1478 . Recruitment of LC3 around the parasite was examined at 5 h after challenge with T . gondii .

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A, hmCD40 mHEVc expressing LC3-EGFP were treated with or without CD154 followed by challenge with T. gondii-RFP in the presence or absence of EGF (50 ng/ml) or AG1478 (1 µM). LC3 accumulation around T. gondii was assessed by immunofluorescence. B, mHEVc-LC3-EGFP cells were infected with T. gondii-T. gondii. Cells were treated with vehicle or rapamycin (1 µM) 2 h after challenge with T. gondii in the presence or absent of EGF or AG1478. Recruitment of LC3 around the parasite was examined at 5 h after challenge with T. gondii.


. C , hmCD40 mHEVc - LC3 - EGFP cells were treated with or without CD154 followed by challenged with WT , MIC1 ko , MIC1 ko + MIC1 , MIC3 ko , MIC3 ko + MIC3 , MIC1 - 3 ko , MIC1 - 3 ko + MIC1 - 3 tachyzoites . LC3 accumulation around the parasite was examined by fluorescence microscopy 5 h post - challenge . D , hmCD40 mHEVc - LC3 - EGFP treated with or without CD154 were challenged with WT , MIC1 ko , MIC1 ko + MIC1 , MIC3 ko , MIC3 ko + MIC3 , MIC1 - 3 ko or MIC1 - 3 ko + MIC1 - 3 tachyzoites . Parasite load was examined 24 h post - challenge .

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. C, hmCD40 mHEVc-LC3-EGFP cells were treated with or without CD154 followed by challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko, MIC1-3 ko+MIC1-3 tachyzoites. LC3 accumulation around the parasite was examined by fluorescence microscopy 5 h post-challenge. D, hmCD40 mHEVc-LC3-EGFP treated with or without CD154 were challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko or MIC1-3 ko+MIC1-3 tachyzoites. Parasite load was examined 24 h post-challenge.


E , HBMEC cells were challenged with WT , MIC1 ko , MIC3 ko , MIC1 - 3 ko tachyzoites followed by treatment with vehicle or rapamycin . Parasite load was examined 24 h post - challenge . F , HBMEC treated with or without CD154 or IFN - γ / TNF - α were challenged with WT , MIC1 ko , MIC3 ko , MIC1 - 3 ko tachyzoites . Monolayers were examined microscopically at 24 h post - challenge .

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

E, HBMEC cells were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites followed by treatment with vehicle or rapamycin. Parasite load was examined 24 h post-challenge. F, HBMEC treated with or without CD154 or IFN-γ/TNF-α were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites. Monolayers were examined microscopically at 24 h post-challenge.


G , hmCD40 mHEVc cells treated with or without CD154 were infected with either WT or MIC1 - 3 ko tachyzoites in the presence of complete medium ( CM ) alone or medium plus MIC3 , MIC4 , M2AP or MIC6 ( all 10 nM ) . Parasite load was determined microscopically 24 h post challenge . Results shown are representative of 3 - 4 independent experiments .

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

G, hmCD40 mHEVc cells treated with or without CD154 were infected with either WT or MIC1-3 ko tachyzoites in the presence of complete medium (CM) alone or medium plus MIC3, MIC4, M2AP or MIC6 (all 10 nM). Parasite load was determined microscopically 24 h post challenge. Results shown are representative of 3-4 independent experiments.


Size exclusion chromatography profiles of recombinant proteins used in this study . AU : arbitrary units ; WT : wild type ; FL : full - length .

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

Size exclusion chromatography profiles of recombinant proteins used in this study. AU: arbitrary units; WT: wild type; FL: full-length.


Analysis of the interaction between CtUba4 and GST - CtUrm1 ( ratio 2 : 1 or 0 . 5 : 1 when marked with an asterisk ) in the presence of nucleotide derivates by GST pull - down resolved by SDS - PAGE and visualized with Coomassie stain .

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

Analysis of the interaction between CtUba4 and GST-CtUrm1 (ratio 2:1 or 0.5:1 when marked with an asterisk) in the presence of nucleotide derivates by GST pull-down resolved by SDS-PAGE and visualized with Coomassie stain.


Functional analyses of ScUba4 mutant yeast using APM - gel retardation ( top ) and viability in response to rapamycin ( bottom ) . PAGE was supplemented with APM to retard the migration of thiolated tRNAs and allow their visualization by northern blot using an anti - tRNA < sub > UU < em > C < / em > < / sub > < sup > Gl < em > u < / em > < / sup > ; probe . APM : ( [ N - Acryloyl - amino ] phenyl ) mercuric chloride ; YPD : yeast extract peptone dextrose . All residue numbering follows the CtUba4 sequence , but the respective ScUba4 numbering is added in subscript .

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

Functional analyses of ScUba4 mutant yeast using APM-gel retardation (top) and viability in response to rapamycin (bottom). PAGE was supplemented with APM to retard the migration of thiolated tRNAs and allow their visualization by northern blot using an anti-tRNAUUCGlu; probe. APM: ([N-Acryloyl-amino]phenyl)mercuric chloride; YPD: yeast extract peptone dextrose. All residue numbering follows the CtUba4 sequence, but the respective ScUba4 numbering is added in subscript.


Interaction analysis of WT and mutated CtUba4 with GST - CtUrm1 by GST pull - down in the presence of 1mM ATP . CtUba4C202K covalently linked to GST - CtUrm1 is marked by an asterisk .

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

Interaction analysis of WT and mutated CtUba4 with GST-CtUrm1 by GST pull-down in the presence of 1mM ATP. CtUba4C202K covalently linked to GST-CtUrm1 is marked by an asterisk.


Analysis of covalent adduct formation between CtUba4 WT or C202 mutants and carboxylated ( - OH ) or thiocarboxylated ( - SH ) CtUrm1 in the presence or absence of TBH , DTT , TCEP and HA , respectively . ATP : Adenosine triphosphate TBH : tert - Butyl hydroperoxide HA : Hydroxylamine ; DTT : 1 , 4 - Dithiothreitol ; TCEP : Tris ( 2 - carboxyethyl ) phosphine .

Analysis of covalent adduct formation between CtUba4 WT or C202 mutants and carboxylated (-OH) or thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH, DTT, TCEP and HA, respectively. ATP: Adenosine triphosphate TBH: tert-Butyl hydroperoxide HA: Hydroxylamine; DTT: 1,4-Dithiothreitol; TCEP: Tris(2-carboxyethyl)phosphine.


Analysis of covalent adduct formation between CtUba4 WT / cysteine mutants and CtUrm1 - COSH in the presence of oxidizing agents . HP : Hydrogen peroxide ; D : Diamide .

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

Analysis of covalent adduct formation between CtUba4 WT/cysteine mutants and CtUrm1-COSH in the presence of oxidizing agents. HP: Hydrogen peroxide; D: Diamide.


Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin .

Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin.


MS / MS spectrum of the K202 - ε - GGH containing peptide from CtUba4C202K . A representative annotated fragmentation spectrum is shown with the b - and y - ions marked in red and blue , respectively . The precursor ion at m / z 591 , 98 is labelled in green . The peptide sequence is shown at the top with the collision - induced fragmentation pattern . The Urm1 conjugation site was identified by detection of the GGH remnant motif generated by chymotryptic digestion .

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

MS/MS spectrum of the K202-ε-GGH containing peptide from CtUba4C202K. A representative annotated fragmentation spectrum is shown with the b- and y-ions marked in red and blue, respectively. The precursor ion at m/z 591,98 is labelled in green. The peptide sequence is shown at the top with the collision-induced fragmentation pattern. The Urm1 conjugation site was identified by detection of the GGH remnant motif generated by chymotryptic digestion.


Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated ( - SH ) CtUrm1 in the presence or absence of TBH and HA , respectively .

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

Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH and HA, respectively.


( A ) Genome - wide ubiquitin binding profiles identify numerous regions of H2B - ubiquitylation in WT cells and distinct sites of non - H2B ubiquitylation ( ' ubiquitin - hotspot ' , ub - hotspot , ub - HS ) which persist in h2b - K123R and rad6 ∆ strains , and increase in cdc48 mutants ( cdc48 - 6 ) . A 90 kb stretch of chromosome XIII ( ChrXIII ) is depicted . Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody ( see also Appendix Fig . S1A ) and enriched DNA was analyzed on NimbleGen arrays ( Chip - chip ) . DNA from non - specific IgG - ChIP experiments served as background control . Significantly enriched regions are marked by bars above the respective ChIP - chip tracks and are summarized in Dataset EV1 . Data represent means from two independent replicates , except for ubiquitin ( FK2 ) in rad6 ∆ and IgG - ChIP in WT ( n = 1 ) . All experiments , including those using cdc48 - 6 and other temperature - sensitive ( ts ) alleles , were performed at 30 ° C ( semipermissive temperature for ts - alleles ) unless stated otherwise .

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

(A) Genome-wide ubiquitin binding profiles identify numerous regions of H2B-ubiquitylation in WT cells and distinct sites of non-H2B ubiquitylation ('ubiquitin-hotspot', ub-hotspot, ub-HS) which persist in h2b-K123R and rad6∆ strains, and increase in cdc48 mutants (cdc48-6). A 90 kb stretch of chromosome XIII (ChrXIII) is depicted. Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody (see also Appendix Fig. S1A) and enriched DNA was analyzed on NimbleGen arrays (Chip-chip). DNA from non-specific IgG-ChIP experiments served as background control. Significantly enriched regions are marked by bars above the respective ChIP-chip tracks and are summarized in Dataset EV1. Data represent means from two independent replicates, except for ubiquitin (FK2) in rad6∆ and IgG-ChIP in WT (n = 1). All experiments, including those using cdc48-6 and other temperature-sensitive (ts) alleles, were performed at 30°C (semipermissive temperature for ts-alleles) unless stated otherwise.


( B ) Seven ub - HSs show strong correlation between ubiquitin binding and Slx8 enrichment ( Slx8 - 9myc ChIP ) . 16 - kb - windows of the indicated regions centered around the ub - HSs are depicted for ubiquitin and Slx8 binding profiles . Ubiquitin ( FK2 ) data for WT and cdc48 - 6 are from the same experiment as depicted in ( A ) . Data represent means from two independent replicates .

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

(B) Seven ub-HSs show strong correlation between ubiquitin binding and Slx8 enrichment (Slx8-9myc ChIP). 16-kb-windows of the indicated regions centered around the ub-HSs are depicted for ubiquitin and Slx8 binding profiles. Ubiquitin (FK2) data for WT and cdc48-6 are from the same experiment as depicted in (A). Data represent means from two independent replicates.


( D ) Slx8 and Slx5 are recruited to ub - HSs . DNA from Slx8 - 9myc ( top ) or 3HA - Slx5 ( bottom ) ChIP experiments of the indicated strains was analyzed by quantitative real - time PCR ( ChIP - qPCR ) at selected ub - HSs . Data represent mean ± standard deviation ( SD , n = 2 ) . Consistent with a previous study , we did not observe any Slx8 enrichment at centromeres ( van de Pasch et al , 2013 ) , however , we note that we could not confirm the reported centromere binding of Slx5 ( see also Appendix Fig . S2E ) .

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

(D) Slx8 and Slx5 are recruited to ub-HSs. DNA from Slx8-9myc (top) or 3HA-Slx5 (bottom) ChIP experiments of the indicated strains was analyzed by quantitative real-time PCR (ChIP-qPCR) at selected ub-HSs. Data represent mean ± standard deviation (SD, n = 2). Consistent with a previous study, we did not observe any Slx8 enrichment at centromeres (van de Pasch et al, 2013), however, we note that we could not confirm the reported centromere binding of Slx5 (see also Appendix Fig. S2E).


( E ) K48 - linked ubiquitin chains accumulate in a cdc48 mutant ( cdc48 - 3 ) . Ub - K48 ChIP followed by qPCR for same loci as in ( D ) are depicted . See also Fig . EV1C - D . Data represent means ± SD ( n = 2 ) .

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

(E) K48-linked ubiquitin chains accumulate in a cdc48 mutant (cdc48-3). Ub-K48 ChIP followed by qPCR for same loci as in (D) are depicted. See also Fig. EV1C-D. Data represent means ± SD (n = 2).


( F ) Slx5 and Slx8 are required for ub - HS formation . Ub - K48 ChIP - qPCR in strains lacking one subunit of the Slx5 / Slx8 complex ( slx5 ∆ , slx8 ∆ ) . Data represent means ± SD ( n = 3 ) .

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

(F) Slx5 and Slx8 are required for ub-HS formation. Ub-K48 ChIP-qPCR in strains lacking one subunit of the Slx5/Slx8 complex (slx5∆, slx8∆). Data represent means ± SD (n = 3).


( G ) Ufd1 and Npl4 act in concert with Cdc48 to remove ubiquitylated proteins from ub - HS sites . Ub - K48 ChIP for the indicated strains grown at the semi - permissive temperature of 30 ° C . Data represent means ± SD ( n = 2 ) .

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

(G) Ufd1 and Npl4 act in concert with Cdc48 to remove ubiquitylated proteins from ub-HS sites. Ub-K48 ChIP for the indicated strains grown at the semi-permissive temperature of 30°C. Data represent means ± SD (n = 2).


B ) A 39 bp fragment of ub - HS4 is sufficient to drive ectopic ub - HS formation . Fragments of ub - HS4 were integrated ectopically and ub - K48 ChIP - qPCR was performed for the endogenous ub - HS5 and the ectopic ub - HS4 fragments as depicted in ( A ) . contr . : control , empty YI128 - vector was integrated at LEU2 . Experiments were performed in cdc48 ‑ 6 background . Data represent means ± SD ( n = 2 ) . Experimental mapping and bioinformatic prediction identify a similar ub - HS - motif . Comparison between the experimentally mapped ub - HS4 F7 ( B ) and the consensus motif of all ub - HSs identified by the MEME software .

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

B) A 39 bp fragment of ub-HS4 is sufficient to drive ectopic ub-HS formation. Fragments of ub-HS4 were integrated ectopically and ub-K48 ChIP-qPCR was performed for the endogenous ub-HS5 and the ectopic ub-HS4 fragments as depicted in (A). contr.: control, empty YI128-vector was integrated at LEU2. Experiments were performed in cdc48‑6 background. Data represent means ± SD (n = 2). Experimental mapping and bioinformatic prediction identify a similar ub-HS-motif. Comparison between the experimentally mapped ub-HS4 F7 (B) and the consensus motif of all ub-HSs identified by the MEME software.


E ) Euc1 binds to the ub - HS - motif in a Y1H assay . Gal4 - AD - or Gal4 - AD - Euc1 - encoding plasmids were transformed into a Y1H reporter strain as described in ( D ) . Serial dilutions were spotted on control plates and plates lacking histidine with 20mM 3 - amino - triazole ( 3AT ) . Cells were grown at 30 ° C for 3 days .

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

E) Euc1 binds to the ub-HS-motif in a Y1H assay. Gal4-AD- or Gal4-AD-Euc1-encoding plasmids were transformed into a Y1H reporter strain as described in (D). Serial dilutions were spotted on control plates and plates lacking histidine with 20mM 3-amino-triazole (3AT). Cells were grown at 30°C for 3 days.


F ) Euc1 is required for formation of ub - HSs . ChIP with ub - K48 specific antibodies was performed for the indicated strains and enriched DNA was analyzed by qPCR . Data represent means ± SD ( n = 4 ) .

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

F) Euc1 is required for formation of ub-HSs. ChIP with ub-K48 specific antibodies was performed for the indicated strains and enriched DNA was analyzed by qPCR. Data represent means ± SD (n = 4).


Euc1 binds to endogenous ub - HSs . Genome - wide binding profiles of K48 - linked ubiquitin chains were obtained in ChIP - chip experiments as described in Fig . 1A . Euc1 - ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292 - 462 . Data represent means from two independent replicates .

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

Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of K48-linked ubiquitin chains were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates.


Euc1 binds to endogenous ub - HSs . Genome - wide binding profiles of Euc1 were obtained in ChIP - chip experiments as described in Fig . 1A . Euc1 - ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292 - 462 . Data represent means from two independent replicates .

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

Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of Euc1 were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates.


( C ) Euc1 can induce transactivation via its N - terminal 30 amino acids . Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig . 2D and serial dilutions were spotted on control or selective media to test HIS3 - activation . Cells were grown at 30 ° C for 3 days .

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

(C) Euc1 can induce transactivation via its N-terminal 30 amino acids. Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig. 2D and serial dilutions were spotted on control or selective media to test HIS3-activation. Cells were grown at 30°C for 3 days.


( D ) Quantification of HIS3 mRNA levels from strains used in ( C ) . Cells were grown in liquid media with selection for the transformed plasmids ( SC - Leu ) , harvested in logarithmic growth phase and total mRNA was prepared . After reverse transcription , HIS3 mRNA levels were quantified using qPCR ( RT - qPCR ) , normalized first to ACT1 mRNA and then to the empty vector control strain . Data represent means ± SD ( n = 4 ) . p = 2 . 43 * 10 - 5 ( Student ' s t - test ) .

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

(D) Quantification of HIS3 mRNA levels from strains used in (C). Cells were grown in liquid media with selection for the transformed plasmids (SC-Leu), harvested in logarithmic growth phase and total mRNA was prepared. After reverse transcription, HIS3 mRNA levels were quantified using qPCR (RT-qPCR), normalized first to ACT1 mRNA and then to the empty vector control strain. Data represent means ± SD (n = 4). p = 2.43*10-5 (Student's t-test).


( A ) Euc1 interacts with SUMO pathway proteins in a yeast two - hybrid ( Y2H ) assay . A Y2H reporter strain ( PJ69 - 7a ) was transformed with Gal4 DNA - binding domain ( BD ) and Gal4 activation domain ( AD ) fusion constructs in the indicated combinations . AD - SUMO - GG can be conjugated to SUMOylation substrates , while AD - SUMO - AA is conjugation - deficient . Cells were spotted on control media or selective media ( - His ) and grown for 3 and 6 days , respectively .

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

(A) Euc1 interacts with SUMO pathway proteins in a yeast two-hybrid (Y2H) assay. A Y2H reporter strain (PJ69-7a) was transformed with Gal4 DNA-binding domain (BD) and Gal4 activation domain (AD) fusion constructs in the indicated combinations. AD-SUMO-GG can be conjugated to SUMOylation substrates, while AD-SUMO-AA is conjugation-deficient. Cells were spotted on control media or selective media (- His) and grown for 3 and 6 days, respectively.


( B ) Euc1 is SUMOylated by Ubc9 , Siz1 and Siz2 on K231 . Euc1 was immunoprecipitated from the indicated strains and eluates were probed by WB against SUMO ( top ) and Euc1 ( bottom ) . An up - shifted band corresponding to Euc1SUMO was detected with both antibodies . Asterisks denote non - specific bands .

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

(B) Euc1 is SUMOylated by Ubc9, Siz1 and Siz2 on K231. Euc1 was immunoprecipitated from the indicated strains and eluates were probed by WB against SUMO (top) and Euc1 (bottom). An up-shifted band corresponding to Euc1SUMO was detected with both antibodies. Asterisks denote non-specific bands.


( C ) Euc1 is predominantly monoSUMOylated on lysine 231 . Denaturing NiNTA - pulldowns ( NiNTA - PD ) with strains expressing His - tagged SUMO ( HisSUMO ) as indicated and 3FLAGEuc1 constructs under the control of an ADH promoter . Covalently SUMO - modified proteins were enriched and eluates probed with a FLAG antibody to visualize SUMOylated Euc1 . Eluates were probed for SUMO to control for equal pull - down , Pgk1 was probed as input control . Euc1 - KR denotes the K231R mutation here and hereafter .

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(C) Euc1 is predominantly monoSUMOylated on lysine 231. Denaturing NiNTA-pulldowns (NiNTA-PD) with strains expressing His-tagged SUMO (HisSUMO) as indicated and 3FLAGEuc1 constructs under the control of an ADH promoter. Covalently SUMO-modified proteins were enriched and eluates probed with a FLAG antibody to visualize SUMOylated Euc1. Eluates were probed for SUMO to control for equal pull-down, Pgk1 was probed as input control. Euc1-KR denotes the K231R mutation here and hereafter.


( D ) Euc1 SUMOylation is required for ub - HS formation . Ub - K48 ChIP quantified by qPCR for the indicated strains . The ubx5 ∆ background was used to enhance the ubiquitin signal , similar results were obtained in a WT background . Data represent means ± SD ( n = 3 ) .

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(D) Euc1 SUMOylation is required for ub-HS formation. Ub-K48 ChIP quantified by qPCR for the indicated strains. The ubx5∆ background was used to enhance the ubiquitin signal, similar results were obtained in a WT background. Data represent means ± SD (n = 3).


( E ) Euc1 ubiquitylation depends on Slx8 and partly on Euc1 SUMOylation . Denaturing NiNTA - PDs as described for ( C ) but with strains expressing His - ubiquitin ( HisUbi ) and 3FLAGEuc1 under the control of the ADH promoter . WBs were developed with a FLAG antibody to probe for 3FLAGEuc1 , a ubiquitin blot served as PD - control and Dpm1 as input control . The slx8 - CD allele carries the C206S , C209S - mutations ( Xie et al , 2007 ) .

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(E) Euc1 ubiquitylation depends on Slx8 and partly on Euc1 SUMOylation. Denaturing NiNTA-PDs as described for (C) but with strains expressing His-ubiquitin (HisUbi) and 3FLAGEuc1 under the control of the ADH promoter. WBs were developed with a FLAG antibody to probe for 3FLAGEuc1, a ubiquitin blot served as PD-control and Dpm1 as input control. The slx8-CD allele carries the C206S, C209S-mutations (Xie et al, 2007).


SUMOylated Euc1 is required to recruit Slx5 and Slx8 to ub - HSs . ChIP against Slx5 in the indicated genetic backgrounds were quantified by qPCR . Note that Slx5 is still recruited in the absence of Slx8 ( 3HA - Slx5 slx8 ∆ ) , but not vice versa ( Slx8 - 9myc slx5 ∆ ) . Data for WT and 3HA - Slx5 in ( F ) and

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SUMOylated Euc1 is required to recruit Slx5 and Slx8 to ub-HSs. ChIP against Slx5 in the indicated genetic backgrounds were quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA-Slx5 slx8∆), but not vice versa (Slx8-9myc slx5∆). Data for WT and 3HA-Slx5 in (F) and WT


SUMOylated Euc1 is required to recruit Slx8 to ub - HSs . ChIP against Slx8 in the indicated genetic backgrounds were quantified by qPCR . Note that Slx5 is still recruited in the absence of Slx8 ( 3HA - Slx5 slx8 ∆ ) , but not vice versa ( Slx8 - 9myc slx5 ∆ ) . Data for WT and Slx8 - 9myc in ( G )

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SUMOylated Euc1 is required to recruit Slx8 to ub-HSs. ChIP against Slx8 in the indicated genetic backgrounds were quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA-Slx5 slx8∆), but not vice versa (Slx8-9myc slx5∆). Data for WT and Slx8-9myc in (G)


( A ) Euc181 - 183 binds to Slx5 in Y2H assays . Y2H assay performed as in Fig . 4A . Serial dilutions were spotted for each plasmid combination and cells were grown at 30 ° C for 2 days . The Slx5 - RING ∆ construct is deleted for the Slx5 C - terminus from beginning of the RING - domain ( ∆ aa 488 - 619 ) . See also Fig . EV3B .

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(A) Euc181-183 binds to Slx5 in Y2H assays. Y2H assay performed as in Fig. 4A. Serial dilutions were spotted for each plasmid combination and cells were grown at 30°C for 2 days. The Slx5-RING∆ construct is deleted for the Slx5 C-terminus from beginning of the RING-domain (∆aa 488-619). See also Fig. EV3B.


( B ) Euc1 binds Slx5 in vitro . Recombinant purified GST or GSTEuc1 were used in GST pull - down assays to co - precipitate recombinant 6HisSlx5 .

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(B) Euc1 binds Slx5 in vitro. Recombinant purified GST or GSTEuc1 were used in GST pull-down assays to co-precipitate recombinant 6HisSlx5.


( C ) Euc1 binds to Slx5 in vivo . Cell lysates from an euc1 ∆ strain expressing the indicated 3FLAGEuc1 constructs from plasmids ( under EUC1 promoter ) were subjected to immunoprecipitation using anti - FLAG beads . IP - eluates were probed by WB with Slx5 , SUMO and Euc1 antibodies , inputs were probed with Slx5 , FLAG and Dpm1 antibodies ( top to bottom ) . Note that the Euc1 - Slx5 interaction is independent of the Slx8 ligase activity ( slx8 - CD , lanes 5 - 8 ) .

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(C) Euc1 binds to Slx5 in vivo. Cell lysates from an euc1∆ strain expressing the indicated 3FLAGEuc1 constructs from plasmids (under EUC1 promoter) were subjected to immunoprecipitation using anti-FLAG beads. IP-eluates were probed by WB with Slx5, SUMO and Euc1 antibodies, inputs were probed with Slx5, FLAG and Dpm1 antibodies (top to bottom). Note that the Euc1-Slx5 interaction is independent of the Slx8 ligase activity (slx8-CD, lanes 5-8).


( D ) Euc1 Slx5 - binding mutants ( SBM1 , SBM2 ) affect Euc1 - Slx5 interaction in Y2H assays . AD - Euc181 - 183 constructs harboring mutations in the region required for Slx5 binding were probed for interaction with BD - Slx5 constructs as described in Fig . 4A . Mutations : SBM1 : aa 139 - 143 ENQKK > ANAAA , SBM2 : aa 162 - 165 KEVF > AAAA . Serial dilutions were spotted and cells were grown at 30 ° C for 2 days . See Appendix Fig . S6A for expression levels .

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(D) Euc1 Slx5-binding mutants (SBM1, SBM2) affect Euc1-Slx5 interaction in Y2H assays. AD-Euc181-183 constructs harboring mutations in the region required for Slx5 binding were probed for interaction with BD-Slx5 constructs as described in Fig. 4A. Mutations: SBM1: aa 139-143 ENQKK>ANAAA, SBM2: aa 162-165 KEVF>AAAA. Serial dilutions were spotted and cells were grown at 30°C for 2 days. See Appendix Fig. S6A for expression levels.


( E ) Euc1 - SBM constructs show defective Slx5 binding in vivo . Mutations described in ( D ) were introduced into full - length Euc1 constructs ( with or without the K231R mutation ) and FLAG IPs were performed as described in ( C ) . WB analysis showed strong Slx5 - binding defects for the SBM1 / SBM2 and SBM1 + 2 constructs ( top panel , immunoprecipitated Slx5 ) . WBs were probed as in ( C ) .

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(E) Euc1-SBM constructs show defective Slx5 binding in vivo. Mutations described in (D) were introduced into full-length Euc1 constructs (with or without the K231R mutation) and FLAG IPs were performed as described in (C). WB analysis showed strong Slx5-binding defects for the SBM1/SBM2 and SBM1+2 constructs (top panel, immunoprecipitated Slx5). WBs were probed as in (C).


( F ) The Slx5 middle domain ( Slx5 - Md ) is required for interaction with Euc1 . Y2H assays with AD - Euc181 - 183 and BD - Slx5 constructs . Slx5 - Md : aa 201 - 335 , Slx5 - Md ∆ : ∆ aa 201 - 338 . Serial dilutions were spotted and cells were grown at 30 ° C for 4 days . See also Fig . EV3C .

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

(F) The Slx5 middle domain (Slx5-Md) is required for interaction with Euc1. Y2H assays with AD-Euc181-183 and BD-Slx5 constructs. Slx5-Md: aa 201-335, Slx5-Md∆: ∆aa 201-338. Serial dilutions were spotted and cells were grown at 30°C for 4 days. See also Fig. EV3C.


( G ) Euc1 - SBM constructs show defective binding to the Slx5 - Md . Y2H assay with the indicated constructs as in ( D ) . See Appendix Fig . S6A for expression controls .

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(G) Euc1-SBM constructs show defective binding to the Slx5-Md. Y2H assay with the indicated constructs as in (D). See Appendix Fig. S6A for expression controls.


( A ) Euc1 Slx5 - binding mutations impair Euc1 ubiquitylation . His - ubiquitin - modified proteins were enriched by denaturing NiNTA - PDs as described in Fig . 4E . 3FLAG - tagged Euc1 constructs were expressed from plasmids under the control of the ADH promoter . NiNTA - PD eluates were probed with FLAG and ubiquitin ( P4D1 ) antibodies . Whole cell lysates ( input ) were probed with FLAG and Dpm1 antibodies .

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(A) Euc1 Slx5-binding mutations impair Euc1 ubiquitylation. His-ubiquitin-modified proteins were enriched by denaturing NiNTA-PDs as described in Fig. 4E. 3FLAG-tagged Euc1 constructs were expressed from plasmids under the control of the ADH promoter. NiNTA-PD eluates were probed with FLAG and ubiquitin (P4D1) antibodies. Whole cell lysates (input) were probed with FLAG and Dpm1 antibodies.


( B ) The Slx5 - SIMs and Slx5 - Md are required for Euc1 ubiquitylation . NiNTA - PDs for His - ubiquitin as in ( A ) . All strains expressed wild - type 3FLAGEuc1 from plasmids under the ADH promoter and His - ubiquitin . Slx5 constructs were expressed from plasmids under control of the endogenous promoter . WBs were probed as described in ( A ) , Slx5 - levels were probed using an HA antibody . Asterisk denotes a non - specific band . Slx5 - SIM * : SIMs 1 - 4 were mutated as described ( Xie et al , 2010 ) , for SIM5 , aa 477 - 479 ( IIV ) were mutated to alanines . To avoid possible mislocalization by deletion of the Slx5 - Md , which overlaps with a putative NLS ( Westerbeck et al , 2014 ) , an N - terminal NLS was fused to all constructs . See Appendix Fig . S7C for HU complementation .

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

(B) The Slx5-SIMs and Slx5-Md are required for Euc1 ubiquitylation. NiNTA-PDs for His-ubiquitin as in (A). All strains expressed wild-type 3FLAGEuc1 from plasmids under the ADH promoter and His-ubiquitin. Slx5 constructs were expressed from plasmids under control of the endogenous promoter. WBs were probed as described in (A), Slx5-levels were probed using an HA antibody. Asterisk denotes a non-specific band. Slx5-SIM*: SIMs 1-4 were mutated as described (Xie et al, 2010), for SIM5, aa 477-479 (IIV) were mutated to alanines. To avoid possible mislocalization by deletion of the Slx5-Md, which overlaps with a putative NLS (Westerbeck et al, 2014), an N-terminal NLS was fused to all constructs. See Appendix Fig. S7C for HU complementation.


( C ) Euc1 Slx5 - binding mutations ( SBM1 / SBM2 ) reduce / abolish ub - HSs and recruitment of Slx5 to ub - HSs . ChIP - qPCR for ub - K48 ( top ) or Slx5 ( using Slx5 antibody , bottom ) in euc1 ∆ ubx5 ∆ cells expressing the indicated constructs from plasmids under the control of the EUC1 promoter . Data represent means ± SD ( n = 3 ) . See also Appendix Fig . S7E - G .

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(C) Euc1 Slx5-binding mutations (SBM1/SBM2) reduce/abolish ub-HSs and recruitment of Slx5 to ub-HSs. ChIP-qPCR for ub-K48 (top) or Slx5 (using Slx5 antibody, bottom) in euc1∆ ubx5∆ cells expressing the indicated constructs from plasmids under the control of the EUC1 promoter. Data represent means ± SD (n = 3). See also Appendix Fig. S7E-G.


( D ) Slx5 - SIMs and the Slx5 - Md are required for the formation of ub - HSs and recruitment of Slx5 . ChIP - qPCR for ub - K48 ( left ) or Slx5 ( anti - HA ChIP , right ) in ubx5 ∆ cells or slx5 ∆ ubx5 ∆ cells complemented with plasmids expressing the indicated Slx5 - constructs under control of the SLX5 promoter . Data represent means ± SD ( n = 2 ) . See also Appendix Fig . S7H .

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

(D) Slx5-SIMs and the Slx5-Md are required for the formation of ub-HSs and recruitment of Slx5. ChIP-qPCR for ub-K48 (left) or Slx5 (anti-HA ChIP, right) in ubx5∆ cells or slx5∆ ubx5∆ cells complemented with plasmids expressing the indicated Slx5-constructs under control of the SLX5 promoter. Data represent means ± SD (n = 2). See also Appendix Fig. S7H.


( A ) Deletion of EUC1 does not lead to ub - HS adjacent gene deregulation , but rather widespread transcriptome changes . Total RNA isolated from WT and euc1 ∆ cells was polyA - enriched and sequenced ( RNAseq , triplicates ) . Significance testing was based on the Wald test , see Materials and Methods for details . See also Appendix Fig . S8A for quantification of selected transcripts by qPCR .

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(A) Deletion of EUC1 does not lead to ub-HS adjacent gene deregulation, but rather widespread transcriptome changes. Total RNA isolated from WT and euc1∆ cells was polyA-enriched and sequenced (RNAseq, triplicates). Significance testing was based on the Wald test, see Materials and Methods for details. See also Appendix Fig. S8A for quantification of selected transcripts by qPCR.


( B ) RNAseq transcriptome analysis of EUC1 overexpression as in ( A ) . ∆ euc1 cells with pGAL - EUC1 or pEUC1 - EUC1 ( control ) integrated at the URA3 - locus ( YIplac211 ) were grown to mid - log phase and 2 % galactose was added for 3h . See also Appendix Fig . S8E .

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(B) RNAseq transcriptome analysis of EUC1 overexpression as in (A). ∆euc1 cells with pGAL-EUC1 or pEUC1-EUC1 (control) integrated at the URA3-locus (YIplac211) were grown to mid-log phase and 2% galactose was added for 3h. See also Appendix Fig. S8E.


( C ) EUC1 overexpression is toxic at elevated temperatures and upon exposure to the membrane fluidizing drug benzylalcohol . For ( C , D , F ) , serial dilutions of the indicated strains were spotted and grown on YPD control ( or selective media ) plates or under conditions as indicated . See also Fig . EV4A - B .

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(C) EUC1 overexpression is toxic at elevated temperatures and upon exposure to the membrane fluidizing drug benzylalcohol. For (C, D, F), serial dilutions of the indicated strains were spotted and grown on YPD control (or selective media) plates or under conditions as indicated. See also Fig. EV4A-B.


( D ) EUC1 overexpression toxicity depends on DNA binding and transactivation . The indicated EUC1 alleles with endogenous or galactose - inducible promoters were integrated at the URA3 locus ( YIplac211 , euc1 ∆ background ) and spotted on glucose control or galactose plates to induce EUC1 overexpression . See also Appendix Fig . S9A - C .

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(D) EUC1 overexpression toxicity depends on DNA binding and transactivation. The indicated EUC1 alleles with endogenous or galactose-inducible promoters were integrated at the URA3 locus (YIplac211, euc1∆ background) and spotted on glucose control or galactose plates to induce EUC1 overexpression. See also Appendix Fig. S9A-C.


( E ) Aberrant binding of overexpressed EUC1 partially depends on Euc1 DNA binding . ChIP - qPCR of Euc1 after 3 hours galactose induction . Note that IP / input ratios of Euc1 signals are shown , also for the control locus ( contr . , TOS1 promoter ) to highlight Euc1 binding at non - ub - HS sites . STI1 - CIN5 : intergenic region . TEC1 - us : upstream ( promoter ) region . Data represent means ± SD ( n = 3 ) . See also Fig . EV4C - D .

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(E) Aberrant binding of overexpressed EUC1 partially depends on Euc1 DNA binding. ChIP-qPCR of Euc1 after 3 hours galactose induction. Note that IP/input ratios of Euc1 signals are shown, also for the control locus (contr., TOS1 promoter) to highlight Euc1 binding at non-ub-HS sites. STI1-CIN5: intergenic region. TEC1-us: upstream (promoter) region. Data represent means ± SD (n = 3). See also Fig. EV4C-D.


( F ) Simultaneous overexpression of SLX5 and SLX8 rescue EUC1 toxicity . Experiment as in ( D ) , but with concomitant plasmid - borne overexpression ( ADH promoter ) of SLX5 and SLX8 ( bottom panels ) and on media selecting for SLX5 / SLX8 plasmids . See also Fig . EV4E and Appendix Fig . S9D .

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(F) Simultaneous overexpression of SLX5 and SLX8 rescue EUC1 toxicity. Experiment as in (D), but with concomitant plasmid-borne overexpression (ADH promoter) of SLX5 and SLX8 (bottom panels) and on media selecting for SLX5/SLX8 plasmids. See also Fig. EV4E and Appendix Fig. S9D.


EUC1 displays negative genetic interactions with genes involved in general and specific transcriptional regulation ( A ) and in particular with members of the Rpd3L histone deacetylase complex ( B ) . For ( A - D ) , serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used in ( A - D ) contained an extra copy of MED11 ,

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EUC1 displays negative genetic interactions with genes involved in general and specific transcriptional regulation (A) and in particular with members of the Rpd3L histone deacetylase complex (B). For (A-D), serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used in (A-D) contained an extra copy of MED11,


EUC1 displays negative genetic interactions with genes involved in particular with members of the Rpd3L histone deacetylase complex For , serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used in ( A - D ) contained an extra copy of MED11 ,

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

EUC1 displays negative genetic interactions with genes involved in particular with members of the Rpd3L histone deacetylase complex For , serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used in (A-D) contained an extra copy of MED11,


For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used contained an extra copy of MED11 ( C ) Plasmid - borne EUC1 complements genetic interactions with Rpd3L subunits . Empty vector ( - ) or a plasmid encoding EUC1 with its endogenous promoter were transformed in the indicated strains and spotted on selective media .

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

For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used contained an extra copy of MED11 (C) Plasmid-borne EUC1 complements genetic interactions with Rpd3L subunits. Empty vector (-) or a plasmid encoding EUC1 with its endogenous promoter were transformed in the indicated strains and spotted on selective media.


For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used contained an extra copy of MED11 ( D ) EUC1 and Rpd3S act in a common pathway which is redundant with Rpd3L . rco1 ∆ and eaf3 ∆ ( Rpd3S ) show similar phenotypes when paired with sds3 ∆ ( Rpd3L ) as euc1 ∆ and are epistatic with euc1 ∆ .

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

For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used contained an extra copy of MED11 (D) EUC1 and Rpd3S act in a common pathway which is redundant with Rpd3L. rco1∆ and eaf3∆ (Rpd3S) show similar phenotypes when paired with sds3∆ (Rpd3L) as euc1∆ and are epistatic with euc1∆.


( A ) Euc1 is recruited to ub - HSs upon mild heat stress ( 37 ° C , 90 min ) . ChIP - qPCR experiments with strains and conditions as indicated . Data represent means ± SD ( n = 4 ) , see Appendix Fig . S11B for statistical analysis .

O O O B-MEASURED_VAR 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

(A) Euc1 is recruited to ub-HSs upon mild heat stress (37°C, 90 min). ChIP-qPCR experiments with strains and conditions as indicated. Data represent means ± SD (n = 4), see Appendix Fig. S11B for statistical analysis.


The ability to form ub - hotspots is crucial for endogenous EUC1 function . The genetic interaction of EUC1 with SDS3 ( Rpd3L complex ) was rescued with plasmid - borne EUC1 - alleles and serial dilutions were spotted on selective media . ( - ) denotes empty vector .

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

The ability to form ub-hotspots is crucial for endogenous EUC1 function. The genetic interaction of EUC1 with SDS3 (Rpd3L complex) was rescued with plasmid-borne EUC1-alleles and serial dilutions were spotted on selective media. (-) denotes empty vector.


SLX5 also shows negative genetic interactions with SDS3 upon heat stress . Serial dilutions of the indicated strains were spotted on YPD and incubated as indicated . See also Appendix Fig . S12C - D . Note that all strains used in ( A - C ) contained an extra copy of MED11 ,

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

SLX5 also shows negative genetic interactions with SDS3 upon heat stress. Serial dilutions of the indicated strains were spotted on YPD and incubated as indicated. See also Appendix Fig. S12C-D. Note that all strains used in (A-C) contained an extra copy of MED11,


A . Raw 264 . 7 macrophage cells were treated with DMSO ( 0 . 05 % ) , 1 μM control antisense oligonucleotide ( ASO ) , or Rab10 - directed ASO ( 1 μM ) , for four days . Immunoblots are representative of three independent experiments , with similar results obtained . B . Calculated reduction of Rab10 ( n = 3 biologically independent experiments ) . Each dot in the plot presents one independent experiment . Group are shown . Error bars represent ± SEM . Significance was assessed by one - way ANOVA with Tukey ' s post hoc test , with * * * * representing P < 0 . 0001 .

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

A. Raw 264.7 macrophage cells were treated with DMSO (0.05%), 1 μM control antisense oligonucleotide (ASO), or Rab10-directed ASO (1 μM), for four days. Immunoblots are representative of three independent experiments, with similar results obtained. B. Calculated reduction of Rab10 (n=3 biologically independent experiments). Each dot in the plot presents one independent experiment. Group are shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with **** representing P <0.0001.


C . Raw 264 . 7 cells were treated with the indicated ASO for four days , or 50 μM EIPA ( 5 - [ N - ethyl - N - isopropyl ] amiloride ) for 20 min , prior to incubation for 60 min with fluorescent ( tetramethylrhodamine , TRITC ) - labeled dextran ( 70 kDa , shown as red color ) , transferrin , or IgG - conjugated agarose beads . Representative photomicrographs ( from 30 images analyzed for each condition , from three biologically independent experiments ) are shown . Cells were washed , fixed , and stained with DAPI ( shown as blue color ) . Scale bars show 200 μm . D - F . Relative fluorescent signals were calculated as a percent of control - ASO treated cells to indicate ( D ) dextran or ( E ) transferrin uptake level . ( F ) For quantification of phagocytosis , the average number of IgG - agarose beads internalized were calculated as a percent of control - ASO treated cells . 30 images across three independent experiments were quantified for each condition , with each dot in the plot representing results from one image . The group means were calculated and shown . Error bars represent ± SEM . Significance was assessed by one - way ANOVA with Tukey ' s post hoc test , with * * * representing P < 0 . 0001 , N . S P > 0 . 05 .

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

C. Raw 264.7 cells were treated with the indicated ASO for four days, or 50 μM EIPA (5-[N-ethyl-N-isopropyl]amiloride) for 20 min, prior to incubation for 60 min with fluorescent (tetramethylrhodamine, TRITC)-labeled dextran (70 kDa, shown as red color), transferrin, or IgG-conjugated agarose beads. Representative photomicrographs (from 30 images analyzed for each condition, from three biologically independent experiments) are shown. Cells were washed, fixed, and stained with DAPI (shown as blue color). Scale bars show 200 μm. D-F. Relative fluorescent signals were calculated as a percent of control-ASO treated cells to indicate (D) dextran or (E) transferrin uptake level. (F) For quantification of phagocytosis, the average number of IgG-agarose beads internalized were calculated as a percent of control-ASO treated cells. 30 images across three independent experiments were quantified for each condition, with each dot in the plot representing results from one image. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P<0.0001, N.S P>0.05.


G . Primary mouse bone marrow - derived macrophage cells ( BMDM ) from adult male C57BL / 6J mice were treated with murine cytokines to polarize cells towards a monocytic lineage ( mBMDM ) , dendritic lineage ( mBMDC ) , or microglia - like lineage ( mMicroglia , see Methods section ) . Cells were incubated with TRITC - dextran for 30 min prior to washing , fixing , and staining with DAPI . Representative photomicrographs from 30 images analyzed for each condition from n = 3 biologically independent experiments are shown . Scale bars show 200 μm . H - J . Relative fluorescent signals were calculated as a percent of control - ASO treated cells . 30 images across 3 independent experiments were quantified for each condition , with each dot in the dotted plot representing 1 image quantified . The group means were calculated and shown . Error bars represent ± SEM . Significance was assessed by one - way ANOVA with Tukey ' s post hoc test , with * * * * representing P < 0 . 0001 , N . S P > 0 . 05 .

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

G. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male C57BL/6J mice were treated with murine cytokines to polarize cells towards a monocytic lineage (mBMDM), dendritic lineage (mBMDC), or microglia-like lineage (mMicroglia, see Methods section). Cells were incubated with TRITC-dextran for 30 min prior to washing, fixing, and staining with DAPI. Representative photomicrographs from 30 images analyzed for each condition from n=3 biologically independent experiments are shown. Scale bars show 200 μm. H-J. Relative fluorescent signals were calculated as a percent of control-ASO treated cells. 30 images across 3 independent experiments were quantified for each condition, with each dot in the dotted plot representing 1 image quantified. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with ****representing P <0.0001, N.S P>0.05.


K . Human monocytes purified from venous blood draws from healthy adult male volunteers were treated with human cytokines towards a monocytic lineage ( hMDM ) , dendritic lineage ( hMDC ) , or microglia - like lineage ( hMicroglia ) . Cells were incubated with TRITC - dextran for 30 min prior to washing , fixing , and staining with DAPI . Representative photomicrographs from 30 images analyzed for each condition from n = 3 biologically independent experiments are shown . Scale bars show 200 μm . L - N . Relative fluorescent signals were calculated as a percent of control - ASO treated cells . 30 images across 3 independent experiments were quantified for each condition , with each dot in the plot representing one image quantified . The group means were calculated and shown . Error bars represent ± SEM . Significance was assessed by one - way ANOVA with Tukey ' s post hoc test , with * * * * representing P < 0 . 0001 .

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

K. Human monocytes purified from venous blood draws from healthy adult male volunteers were treated with human cytokines towards a monocytic lineage (hMDM), dendritic lineage (hMDC), or microglia-like lineage (hMicroglia). Cells were incubated with TRITC-dextran for 30 min prior to washing, fixing, and staining with DAPI. Representative photomicrographs from 30 images analyzed for each condition from n=3 biologically independent experiments are shown. Scale bars show 200 μm. L-N. Relative fluorescent signals were calculated as a percent of control-ASO treated cells. 30 images across 3 independent experiments were quantified for each condition, with each dot in the plot representing one image quantified. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with **** representing P <0.0001.


A . Raw 264 . 7 macrophage cells were transfected with FLAG ( N - term ) - Rab10 and eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) , or eGFP ( C - term ) - SARA - Fyve domain ( PI3P marker ) , or eGFP ( C - term ) - PLC - PH domain ( PI ( 4 , 5 ) P2 marker ) , or mkate2 ( N - term ) - 2xTAPP1 - PH domain ( PI ( 3 , 4 ) P2 marker ) . Representative photomicrographs ( from 25 images analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed cells immuno - stained for FLAG - tag ( Rab10 , shown as green ) together with eGFP epifluorescence ( shown as red ) . White bounding boxes show " High Mag " panels that magnify representative individual vesicles . The colocalization of Rab10 and indicated markers in the bounding boxes were analyzed using Image J as described in the method and shown in the " CoLoc " panels . The colocalized pixels are shown in white with the same intensity . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels and " CoLoc " panels . White arrow heads indicate representative Rab10 vesicles positively colocalized with the indicated marker . B . At least 25 cells across three independent experiments were analyzed for each condition ( see Materials and Methods section ) . % of Rab10 vesicles positive with the indicated marker within each cell was quantified . Each dot represents the mean value from one cell analyzed . Group means are given , with error bars representing ± SEM .

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

A. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain (PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker), or mkate2(N-term)-2xTAPP1-PH domain (PI(3,4)P2 marker). Representative photomicrographs (from 25 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). White bounding boxes show "High Mag" panels that magnify representative individual vesicles. The colocalization of Rab10 and indicated markers in the bounding boxes were analyzed using Image J as described in the method and shown in the "CoLoc" panels. The colocalized pixels are shown in white with the same intensity. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate representative Rab10 vesicles positively colocalized with the indicated marker. B. At least 25 cells across three independent experiments were analyzed for each condition (see Materials and Methods section). % of Rab10 vesicles positive with the indicated marker within each cell was quantified. Each dot represents the mean value from one cell analyzed. Group means are given, with error bars representing ± SEM.


C . Raw 264 . 7 macrophage cells were transfected with FLAG ( N - term ) - Rab10 and eGFP ( C - term ) - Akt - PH domain ( PI ( 3 , 4 , 5 ) P3 marker ) and treated with wortmannin ( 1 μM ) for one hour . Representative photomicrographs ( from 25 cells analyzed for each condition from n = 3 biologically independent experiments ) are shown from fixed cells immuno - stained for FLAG - tag ( Rab10 , shown as green ) together with eGFP epifluorescence ( shown as red ) . Scale bars are 10 μm , or 1 μm in " Hig Mag " panel . D . Percent of cells treated with or without wortmannin harboring Rab10 vesicles was calculated from > 30 cells across five independent experiments . Each dot on the plot represents mean values from one experiment . Error bars represent ± SEM and significance was assessed by Mann - Whitney test ( owing to non - normal distributions ) with * * indicating P < 0 . 01 .

O O O O O O O O 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 I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-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 B-CONTROLLED_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 O O O O 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. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker) and treated with wortmannin (1 μM) for one hour. Representative photomicrographs (from 25 cells analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). Scale bars are 10 μm, or 1 μm in "Hig Mag" panel. D. Percent of cells treated with or without wortmannin harboring Rab10 vesicles was calculated from >30 cells across five independent experiments. Each dot on the plot represents mean values from one experiment. Error bars represent ± SEM and significance was assessed by Mann-Whitney test (owing to non-normal distributions) with ** indicating P<0.01.


E . Raw 264 . 7 cells were co - transfected with eGFP ( N - term ) - Rab10 and mRuby ( N - term ) - Rab5 for 48 hours , with live - cell recordings for ~ 10 minutes . A representative vesicle is shown over the presented time - lapse . Scale bar represents 1 μm . F . Fluorescence intensities of individual vesicles are calculated over time . Dots show mean values calculated from n = 8 recorded vesicles from cells from n = 3 biologically independent experiments . Error bars show ± SEM .

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E. Raw 264.7 cells were co-transfected with eGFP(N-term)-Rab10 and mRuby(N-term)-Rab5 for 48 hours, with live-cell recordings for ~10 minutes. A representative vesicle is shown over the presented time-lapse. Scale bar represents 1 μm. F. Fluorescence intensities of individual vesicles are calculated over time. Dots show mean values calculated from n=8 recorded vesicles from cells from n=3 biologically independent experiments. Error bars show ± SEM.


Raw264 . 7 cells were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . 24 - hours later , cells were incubated with ( A ) TRITC - dextran ( 70 kDa , shown as red signal ) Representative photomicrographs are from 25 cells each analyzed for each condition from n = 3 biologically independent experiments . White bounding boxes are magnified in " High Mag " panels that show individual vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in white . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels and " CoLoc " panels . White arrow heads indicate Rab10 vesicles colocalized with the indicated marker .

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Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (A) TRITC-dextran (70 kDa, shown as red signal) Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.


Raw264 . 7 cells were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . 24 - hours later , cells were incubated with ( B ) DQ - ovalbumin , for 30 min before washing and fixation . Representative photomicrographs are from 25 cells each analyzed for each condition from n = 3 biologically independent experiments . White bounding boxes are magnified in " High Mag " panels that show individual vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in white . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels and " CoLoc " panels . White arrow heads indicate Rab10 vesicles colocalized with the indicated marker .

Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (B) DQ-ovalbumin, for 30 min before washing and fixation. Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.


Raw264 . 7 cells were transfected with plasmids expressing eGFP ( N - term ) - WT - Rab10 or Q68L - Rab10 ( GTP - locked ) . ( C , D ) Cells were further stained for ( C ) EEA1 , or ( D ) Lamp1 , which was detected with Cy5 dye ( show as red signal ) . Representative photomicrographs are from 25 cells each analyzed for each condition from n = 3 biologically independent experiments . White bounding boxes are magnified in " High Mag " panels that show individual vesicles . Positive co - localization signal ( see Methods ) is indicated in bounding boxes labeled as " CoLoc . " Colocalized pixels are shown in white . Scale bars are 10 μm , or 1 μm in " Hig Mag " panels and " CoLoc " panels . White arrow heads indicate Rab10 vesicles colocalized with the indicated marker .

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

Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). (C, D) Cells were further stained for (C) EEA1, or (D) Lamp1, which was detected with Cy5 dye (show as red signal). Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in "High Mag" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as "CoLoc." Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.