( E ) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC . RF or ULK1 siRNA ‐ treated cells were co ‐ transfected with FEZ1 - GFP and FLAG - SCOC , then incubated and immunoprecipitated as in ( C ) . Western blot of lysates for ULK1 , GFP and FLAG , and immunoprecipitates with GFP and FLAG antibodies .

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(E) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC. RF or ULK1 siRNA‐treated cells were co‐transfected with FEZ1-GFP and FLAG-SCOC, then incubated and immunoprecipitated as in (C). Western blot of lysates for ULK1, GFP and FLAG, and immunoprecipitates with GFP and FLAG antibodies.


( F ) siRNA depletion of SCOC reduces FEZ1 - ULK1 complex . HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1 - GFP . FEZ1 - GFP was detected with anti ‐ GFP in both BN - PAGE and SDS - PAGE . Endogenous ULK1 was detected with anti ‐ ULK1 antibody . Arrowheads indicate a FEZ1 - GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex > 1236 kDa also detected in ( D ) in the triple transfection . The 200 KDa complex is sensitive to loss of SCOC and ULK1 .

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(F) siRNA depletion of SCOC reduces FEZ1-ULK1 complex. HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1-GFP. FEZ1-GFP was detected with anti‐GFP in both BN-PAGE and SDS-PAGE. Endogenous ULK1 was detected with anti‐ULK1 antibody. Arrowheads indicate a FEZ1-GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex >1236 kDa also detected in (D) in the triple transfection. The 200 KDa complex is sensitive to loss of SCOC and ULK1.


( G ) SCOC can reduce the interaction of ULK1 with MBP - FEZ1 . Immobilised MBP - FEZ1 was incubated with in vitro 35S ‐ methionine ‐ labelled Myc - ULK1 . Addition of increasing amounts of in vitro translated 35S ‐ methionine SCOC reduced the amount of Myc - ULK1 bound . Top and bottom panels are as described in ( A ) .

(G) SCOC can reduce the interaction of ULK1 with MBP-FEZ1. Immobilised MBP-FEZ1 was incubated with in vitro 35S‐methionine‐labelled Myc-ULK1. Addition of increasing amounts of in vitro translated 35S‐methionine SCOC reduced the amount of Myc-ULK1 bound. Top and bottom panels are as described in (A).


( A ) SCOC interacts with UVRAG . HEK293 cells were transfected with FLAG - SCOC and Myc - UVRAG , lysed and incubated with anti ‐ FLAG ( left ) or anti ‐ Myc ( right ) antibodies . Co ‐ immunoprecipitation of Myc - UVRAG ( left ) and FLAG - SCOC ( right ) was confirmed using anti ‐ Myc and anti ‐ FLAG antibodies for western blotting . Asterisk in ( A - C ) and ( D ) indicates non ‐ specific bands .

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(A) SCOC interacts with UVRAG. HEK293 cells were transfected with FLAG-SCOC and Myc-UVRAG, lysed and incubated with anti‐FLAG (left) or anti‐Myc (right) antibodies. Co‐immunoprecipitation of Myc-UVRAG (left) and FLAG-SCOC (right) was confirmed using anti‐Myc and anti‐FLAG antibodies for western blotting. Asterisk in (A-C) and (D) indicates non‐specific bands.


( B ) Interaction of SCOC with UVRAG is unaffected by FEZ1 - GFP . As in ( A ) , co ‐ immunoprecipitation was preformed in HEK293 cells transfected with FLAG - SCOC , Myc - UVRAG and FEZ1 - GFP . Myc - UVRAG was detected as in ( A ) , FEZ1 - GFP was detected with anti ‐ GFP antibody .

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(B) Interaction of SCOC with UVRAG is unaffected by FEZ1-GFP. As in (A), co‐immunoprecipitation was preformed in HEK293 cells transfected with FLAG-SCOC, Myc-UVRAG and FEZ1-GFP. Myc-UVRAG was detected as in (A), FEZ1-GFP was detected with anti‐GFP antibody.


( C ) The SCOC and UVRAG interaction is reduced by starvation , and this reduction is inhibited by FEZ1 - GFP . HEK293 cells were transfected as in ( B ) and 24 h later either treated with FM or ES for 2 h . FLAG - SCOC or Myc - UVRAG was immunoprecipitated with anti ‐ FLAG or anti ‐ Myc antibodies and the bound proteins were detected with anti ‐ Myc , ‐ GFP or ‐ FLAG antibodies by western blotting . Quantification of the amount of FLAG - SCOC co ‐ immunoprecipitated with Myc - UVRAG in FM or ES , without overexpressed FEZ1 - GFP ( left ) or with overexpressed FEZ1 - GFP ( right ) . Significance was determined using a two ‐ tailed paired t ‐ test ( n = 3 ) .

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(C) The SCOC and UVRAG interaction is reduced by starvation, and this reduction is inhibited by FEZ1-GFP. HEK293 cells were transfected as in (B) and 24 h later either treated with FM or ES for 2 h. FLAG-SCOC or Myc-UVRAG was immunoprecipitated with anti‐FLAG or anti‐Myc antibodies and the bound proteins were detected with anti‐Myc, ‐GFP or ‐FLAG antibodies by western blotting. Quantification of the amount of FLAG-SCOC co‐immunoprecipitated with Myc-UVRAG in FM or ES, without overexpressed FEZ1-GFP (left) or with overexpressed FEZ1-GFP (right). Significance was determined using a two‐tailed paired t‐test (n=3).


( D ) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction . HEK293 cells were transfected with FEZ1 - GFP , GFP or FLAG - SCOC alone or in combination . Top panel input , bottom panel immunoprecipitation with anti ‐ GFP or anti ‐ FLAG . Western blot for endogenous UVRAG , GFP - FEZ1 GFP or FLAG - SCOC as indicated . Arrowhead : endogenous UVRAG .

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(D) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction. HEK293 cells were transfected with FEZ1-GFP, GFP or FLAG-SCOC alone or in combination. Top panel input, bottom panel immunoprecipitation with anti‐GFP or anti‐FLAG. Western blot for endogenous UVRAG, GFP-FEZ1 GFP or FLAG-SCOC as indicated. Arrowhead: endogenous UVRAG.


( E ) UVRAG can interact with both FEZ1 and SCOC in vitro . GST , GST - FEZ1 or GST - SCOC were immobilised and incubated with 35S ‐ labelled Myc - UVRAG as indicated . Arrowheads indicate GST - FEZ1 ( left ) and GST - SCOC ( right ) .

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(E) UVRAG can interact with both FEZ1 and SCOC in vitro. GST, GST-FEZ1 or GST-SCOC were immobilised and incubated with 35S‐labelled Myc-UVRAG as indicated. Arrowheads indicate GST-FEZ1 (left) and GST-SCOC (right).


( F ) Endogenous UVRAG partially colocalises with endogenous FEZ1 in HEK293 cells . Arrows show colocalisation . Scale bar = 10 μm .

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(F) Endogenous UVRAG partially colocalises with endogenous FEZ1 in HEK293 cells. Arrows show colocalisation. Scale bar=10 μm.


( A ) Depletion of endogenous WAC . Anti ‐ WAC and ‐ Actin blot and WAC mRNA levels by qRT - PCR after treatment with RF or individual WAC siRNAs ( W ‐ 01 to W ‐ 04 ) in HEK293 cells . qRT - PCR error bars represent standard deviation of triplicates ( n = 2 ) .

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(A) Depletion of endogenous WAC. Anti‐WAC and ‐Actin blot and WAC mRNA levels by qRT-PCR after treatment with RF or individual WAC siRNAs (W‐01 to W‐04) in HEK293 cells. qRT-PCR error bars represent standard deviation of triplicates (n=2).


( B ) ( B ) WAC is required for autophagy . Anti ‐ ULK1 , ‐ WAC , ‐ Actin and ‐ LC3 blots after siRNA treatment in HEK293 cells and incubation in FM , ES or EL . Representative blot ; quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ tailed paired t ‐ test : RF EL versus siWAC ‐ 03 EL , * * P = 0 . 0079 .

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(B) (B) WAC is required for autophagy. Anti‐ULK1, ‐WAC, ‐Actin and ‐LC3 blots after siRNA treatment in HEK293 cells and incubation in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siWAC‐03 EL, **P=0.0079.


( C ) p62 degradation requires WAC . Anti ‐ p62 and ‐ Actin blot after siRNA treatment of GFP - LC3 ‐ HEK cells in FM or ES for 2 h in duplicate . Bars represent p62 / actin levels normalised to RF FM ; quantification of p62 / actin ; error bars represent s . e . m . ( n = 3 ) : RF FM versus RF ES , * * P = 0 . 0057 ; RF FM versus siWAC ‐ 03 FM , * * P = 0 . 0025 ; RF ES versus siWAC ‐ 03 ES , * P = 0 . 0386 .

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(C) p62 degradation requires WAC. Anti‐p62 and ‐Actin blot after siRNA treatment of GFP-LC3‐HEK cells in FM or ES for 2 h in duplicate. Bars represent p62/actin levels normalised to RF FM; quantification of p62/actin; error bars represent s.e.m. (n=3): RF FM versus RF ES, **P=0.0057; RF FM versus siWAC‐03 FM, **P=0.0025; RF ES versus siWAC‐03 ES, *P=0.0386.


( D ) WAC depletion accelerates degradation of a UPS reporter . Anti ‐ p62 , ‐ GFP , ‐ Actin , ‐ LC3 , ‐ WAC and ‐ Atg16 blots after siRNA treatment in UbG76V - YFP MelJuSo cells and incubation conditions FM , ES or FM with 10 μM MG132 ( MG ) for 2 h . Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC ‐ 03 siRNA treatment ( n = 3 ) and compared with ATG16 siRNA treatment ( n = 2 ) . Error bars represent s . e . m . : RF FM versus siWAC ‐ 03 FM , * * P = 0 . 0082 .

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(D) WAC depletion accelerates degradation of a UPS reporter. Anti‐p62, ‐GFP, ‐Actin, ‐LC3, ‐WAC and ‐Atg16 blots after siRNA treatment in UbG76V-YFP MelJuSo cells and incubation conditions FM, ES or FM with 10 μM MG132 (MG) for 2 h. Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC‐03 siRNA treatment (n=3) and compared with ATG16 siRNA treatment (n=2). Error bars represent s.e.m.: RF FM versus siWAC‐03 FM, **P=0.0082.


( E ) Soluble HttQ25 - CFP degradation is increased after WAC depletion . Anti ‐ WAC , ‐ Actin and ‐ GFP blots after indicated siRNA treatment ( siWAC duplexes ‐ 02 , ‐ 03 , ‐ 04 ) of HttPolyQ ‐ mutant HeLa cell lines and incubation conditions DMSO or DOX ( Doxycyline ) for 3 days . The insoluble fraction was obtained by pelleting the HttQ103 lysates ( see Materials and methods ) . Quantification of normalised average HttQ25 - CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment ; error bar represents s . e . m . ( n = 3 ) : HttQ25 - CFP / actin RF DMSO versus siWAC DMSO , * P = 0 . 0350 ; soluble HttQ103 - CFP / actin RF DMSO versus siWAC DMSO , * P = 0 . 0380 .

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(E) Soluble HttQ25-CFP degradation is increased after WAC depletion. Anti‐WAC, ‐Actin and ‐GFP blots after indicated siRNA treatment (siWAC duplexes ‐02, ‐03, ‐04) of HttPolyQ‐mutant HeLa cell lines and incubation conditions DMSO or DOX (Doxycyline) for 3 days. The insoluble fraction was obtained by pelleting the HttQ103 lysates (see Materials and methods). Quantification of normalised average HttQ25-CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment; error bar represents s.e.m. (n=3): HttQ25-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0350; soluble HttQ103-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0380.


A . Characterization of C . perfringens β - toxin ( CPB ) constructs with different N - termini by gel electrophoretic analysis under denaturing ( A ) conditions . SDS - PAGE gel analyis of CPB samples in cholate ( 1μg per lane ) . CPB samples containing cholate were boiled for 5 min at 95 ° C ( + ) or not ( - ) .

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A. Characterization of C. perfringens β-toxin (CPB) constructs with different N-termini by gel electrophoretic analysis under denaturing (A) conditions. SDS-PAGE gel analyis of CPB samples in cholate (1μg per lane). CPB samples containing cholate were boiled for 5 min at 95°C (+) or not (-).


B . Coomassie stained Blue native PAGE gel ( 4 - 16 % ) of CPB samples ( 10ug per lane ) showing oligomer formation for all different N - termini constructs .

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B. Coomassie stained Blue native PAGE gel (4-16%) of CPB samples (10ug per lane) showing oligomer formation for all different N-termini constructs.


C . Alignment of CPB N - terminus with N - termini of different hemolysins and cryo - EM 2D classification and average of N - terminally tagged WT CPB ( 1 ) , Δ23CPB ( 2 ) , δ - toxin N - terminus Δ23CPB ( 3 ) , C - terminally tagged WT CPB ( 4 ) , Hla N - terminus Δ23CPB ( 5 ) and HlgB N - terminus Δ23CPB ( 6 ) .

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C. Alignment of CPB N-terminus with N-termini of different hemolysins and cryo-EM 2D classification and average of N-terminally tagged WT CPB (1), Δ23CPB (2), δ-toxin N-terminus Δ23CPB (3), C-terminally tagged WT CPB (4), Hla N-terminus Δ23CPB (5) and HlgB N-terminus Δ23CPB (6).


D . Viability of HEK 293FT / CD31 - GFP cells ( transduced with CD31 - GFP ) as a percentage of untreated control cells after incubation with indicated concentrations of toxins ( 24 h , 37 ° C ) . Data ( technical replicates ) are represented as means ( n = 4 ) ± SD .

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D. Viability of HEK 293FT/CD31-GFP cells (transduced with CD31-GFP) as a percentage of untreated control cells after incubation with indicated concentrations of toxins (24 h, 37°C). Data (technical replicates) are represented as means (n = 4) ± SD.


( B ) Cells expressing fluorescently - tagged α2 - tubulin ( atb2 ) were imaged every 5 seconds and the dwell time of ~ 100 individual iMTs recorded within the final 1 . 1 µm of the cell for each strain . Red bars signify the

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(B) Cells expressing fluorescently-tagged α2-tubulin (atb2) were imaged every 5 seconds and the dwell time of ~100 individual iMTs recorded within the final 1.1 µm of the cell for each strain. Red bars signify the mean


( C ) Kymographs showing fluorescently - tagged Klp5 / Klp6 ( Kinesin ) co - imaged with fluorescently - tagged microtubules ( MT ) in the presence ( top panels ) and absence ( bottom panels ) of Mcp1 . Banding on MT , caused by unequal incorporation of fluorescence , illustrates the force exerted on the MT by continued growth into the cell cortex . Dashed yellow line indicates the cell

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(C) Kymographs showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged microtubules (MT) in the presence (top panels) and absence (bottom panels) of Mcp1. Banding on MT, caused by unequal incorporation of fluorescence, illustrates the force exerted on the MT by continued growth into the cell cortex. Dashed yellow line indicates the cell end


( D ) MT growth speed was calculated from kymographs from control ( n = 16 ) and ∆ mcp1 cells ( n = 11 ) , Klp5 / Klp6 walk speed was calculated from multiple individual runs on the MT lattice in control ( n = 44 ) and ∆ mcp1 cells ( n = 32 )

(D) MT growth speed was calculated from kymographs from control (n=16) and ∆mcp1 cells (n=11), Klp5/Klp6 walk speed was calculated from multiple individual runs on the MT lattice in control (n=44) and ∆mcp1 cells (n=32)


( E ) Average intensity of Klp5 / Klp6 at the plus ends of iMTs from multiple kymographs of control ( n = 19 ) or ∆ mcp1 cells ( n = 14 )

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(E) Average intensity of Klp5/Klp6 at the plus ends of iMTs from multiple kymographs of control (n=19) or ∆mcp1 cells (n=14)


( F ) Mixing experiment to compare fluorescently - tagged Klp5 / Klp6 levels between cells either expressing ( blue , closed arrowheads ) or deleted ( red , open arrowhead ) for Mcp1 distinguished by the absence of fluorescently - tagged nuclear envelope protein Cut11 ( left panel ) . Scale bar , 5µm . Box plot ( right panel ) shows quantitated fluorescence values for nuclear levels of Klp5 / Klp6 in control ( n = 44 ) and ∆ mcp1 cells ( n = 45 ) and at the MT plus end in control ( n = 64 ) and ∆ mcp1 cells ( n = 35 ) prior to

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(F) Mixing experiment to compare fluorescently-tagged Klp5/Klp6 levels between cells either expressing (blue, closed arrowheads) or deleted (red, open arrowhead) for Mcp1 distinguished by the absence of fluorescently-tagged nuclear envelope protein Cut11 (left panel). Scale bar, 5µm. Box plot (right panel) shows quantitated fluorescence values for nuclear levels of Klp5/Klp6 in control (n=44) and ∆mcp1 cells (n=45) and at the MT plus end in control (n=64) and ∆mcp1 cells (n=35) prior to shrinkage


( A ) Log phase cells expressing fluorescently - tagged MTs were imaged and the levels of either fluorescently - tagged Klp5 / Klp6 or Tea2 at the plus ends of growing MTs determined . Measurements ( klp5 - mNG klp6 - mNG , n = 305 ; tea2 - GFP , n = 359 ) were plotted against microtubule ( MT ) length and third order polynomial curves fitted to the

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(A) Log phase cells expressing fluorescently-tagged MTs were imaged and the levels of either fluorescently-tagged Klp5/Klp6 or Tea2 at the plus ends of growing MTs determined. Measurements (klp5-mNG klp6-mNG, n=305; tea2-GFP, n=359) were plotted against microtubule (MT) length and third order polynomial curves fitted to the data


( B ) Kymographs showing fluorescently - tagged MTs co - imaged with fluorescently - tagged Tea2 kinesin in control cells ( top panels ) or in the absence of Klp6 ( middle panels ) or Mcp1 ( bottom panels ) . Dashed yellow line indicates the cell

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(B) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Tea2 kinesin in control cells (top panels) or in the absence of Klp6 (middle panels) or Mcp1 (bottom panels). Dashed yellow line indicates the cell end


( C ) Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in ( B ) for control ( n = 16 ) , ∆ klp6 ( n = 13 ) or ∆ mcp1 ( n = 14 ) cells

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(C) Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in (B) for control (n=16), ∆klp6 (n=13) or ∆mcp1 (n=14) cells


( D ) Kymographs showing fluorescently - tagged MTs co - imaged with fluorescently - tagged Klp5 / Klp6 in control ( top panels , repeated from 1C ) or ∆ tea2 cells ( bottom panels ) . Dashed yellow line indicates the cell

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(D) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Klp5/Klp6 in control (top panels, repeated from 1C) or ∆tea2 cells (bottom panels). Dashed yellow line indicates the cell end


( E ) Cells expressing fluorescently - tagged tubulin were imaged every 5 seconds and the dwell time of ~ 100 individual iMTs for each condition recorded within the final 1 . 1 µm of the cell . Red bars signify the

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(E) Cells expressing fluorescently-tagged tubulin were imaged every 5 seconds and the dwell time of ~100 individual iMTs for each condition recorded within the final 1.1 µm of the cell. Red bars signify the mean


( A ) Kymograph ( top left panels ) showing fluorescently - tagged Tea2 ( Kinesin ) co - imaged with fluorescently - tagged Tip1 ( Tip1 - Tdtom ) . Dashed yellow line indicates the position of the cell end . Plots ( bottom left panels ) show both the relative fluorescence intensity and position for Tea2 ( green ) and Tip1 ( magenta ) corresponding to the numbered sections of the kymograph . Scale bar 2µm . Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end for Tea2 ( green ) and Tip1 ( magenta ) over time ( top right ) and the distance of these pixels from the cell end ( bottom right )

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(A) Kymograph (top left panels) showing fluorescently-tagged Tea2 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the position of the cell end. Plots (bottom left panels) show both the relative fluorescence intensity and position for Tea2 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar 2µm. Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end for Tea2 (green) and Tip1 (magenta) over time (top right) and the distance of these pixels from the cell end (bottom right)


( A ) Kymograph ( top left panels ) showing fluorescently - tagged Klp5 / Klp6 ( Kinesin ) co - imaged with fluorescently - tagged Tip1 ( Tip1 - Tdtom ) . Dashed yellow line indicates the cell end . Plots ( bottom left panels ) show both the fluorescence intensity and position of Klp5 / Klp6 ( green ) and Tip1 ( magenta ) corresponding to the numbered sections of the kymograph . Scale bar , 2µm . Intensity of the maximal value pixel at the plus end ( top right panel ) and its position relative to the cell end ( bottom right panel ) for both Klp5 / Klp6 ( green ) and Tip1 ( magenta ) plotted relative to MT shrinkage for this kymograph

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(A) Kymograph (top left panels) showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the cell end. Plots (bottom left panels) show both the fluorescence intensity and position of Klp5/Klp6 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar, 2µm. Intensity of the maximal value pixel at the plus end (top right panel) and its position relative to the cell end (bottom right panel) for both Klp5/Klp6 (green) and Tip1 (magenta) plotted relative to MT shrinkage for this kymograph


A ) The absolute abundance of centrosomal proteins in whole cell lysates , prepared from asynchronous growing cells , was determined using SRM mass spectrometry combined with stable isotope dilution . Copy numbers per cell were measured in three independent experiments . Because not all peptides could be detected in all experiments , histograms show average values determined in those two measurements in which most peptides could be measured reliably ; Results presented are means ± SEM . RPE - 1 ( non - transformed ) cells were compared with various tumor - derived ( U2OS , HeLa , KE37 ) or transformed cells ( HEK293T ) . Note the different scale for γ - tubulin on the y - axis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The absolute abundance of centrosomal proteins in whole cell lysates, prepared from asynchronous growing cells, was determined using SRM mass spectrometry combined with stable isotope dilution. Copy numbers per cell were measured in three independent experiments. Because not all peptides could be detected in all experiments, histograms show average values determined in those two measurements in which most peptides could be measured reliably; Results presented are means ± SEM. RPE-1 (non-transformed) cells were compared with various tumor-derived (U2OS, HeLa, KE37) or transformed cells (HEK293T). Note the different scale for γ-tubulin on the y-axis.


B ) Representative image showing centrosome preparation stained with antibodies to NEDD1 ( green ) and CP110 ( red ) . Bar : 10 μm .

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B) Representative image showing centrosome preparation stained with antibodies to NEDD1 (green) and CP110 (red). Bar: 10 μm.


C ) Purified centrosomes from KE37 cells were analyzed by SRM with stable isotope dilution . Histogram a priori represents relative copy numbers per centrosome ( Results presented are means ± SEM . ) . To predict protein abundance in absolute numbers ( average number of copies / centrosome ) , data were normalized to 1340 γ - tubulin molecules , i . e . the average number of γ - tubulin per centrosome as deduced by combining SRM and EGFP fluorescence measurements ( Figs 4 and 5 ) . Bars for Sas - 6 and STIL are marked in red to indicate that the corresponding values should be approximately doubled to correct for the fact that some 50 % of purified centrosomes are derived from G1 phase cells that mostly lack these two proteins . Note the different scale for γ - tubulin on the y - axis .

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C) Purified centrosomes from KE37 cells were analyzed by SRM with stable isotope dilution. Histogram a priori represents relative copy numbers per centrosome (Results presented are means ± SEM.). To predict protein abundance in absolute numbers (average number of copies/centrosome), data were normalized to 1340 γ-tubulin molecules, i.e. the average number of γ-tubulin per centrosome as deduced by combining SRM and EGFP fluorescence measurements (Figs 4 and 5). Bars for Sas-6 and STIL are marked in red to indicate that the corresponding values should be approximately doubled to correct for the fact that some 50% of purified centrosomes are derived from G1 phase cells that mostly lack these two proteins. Note the different scale for γ-tubulin on the y-axis.


Centrosome preparations from KE37 cells were analyzed by shotgun proteomics and relative protein abundances determined using the iBAQ shotgun proteomics method . The iBAQ values were normalized with the trendline as obtained from the SRM measurements ( Fig EV1 , panel E ) and γ - tubulin was normalized to 1340 copies / centrosome . A subset of centrosomal proteins is shown as described in ( Azimzadeh et al , 2012 ) . Black bars represent the centrosome subset and red bars represent the values obtained by SRM . Results presented are means ± 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Centrosome preparations from KE37 cells were analyzed by shotgun proteomics and relative protein abundances determined using the iBAQ shotgun proteomics method. The iBAQ values were normalized with the trendline as obtained from the SRM measurements (Fig EV1, panel E) and γ-tubulin was normalized to 1340 copies/centrosome. A subset of centrosomal proteins is shown as described in (Azimzadeh et al, 2012). Black bars represent the centrosome subset and red bars represent the values obtained by SRM. Results presented are means ± SEM.


A ) Fluorescence analysis of RPE - 1 cells expressing endogenously tagged γ - tubulin - EGFP . Cells were synchronized in G2 , using RO - 3306 , to allow for a clear distinction of the two centrosomes present at this cell cycle stage . Panels illustrate sum projections of the stacks from an original picture ( left ) , a whole cell mask ( middle ) and a centrosome mask ( right ) ; both masks are based on intensity thresholding . Bar : 10 μm .

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A) Fluorescence analysis of RPE-1 cells expressing endogenously tagged γ-tubulin-EGFP. Cells were synchronized in G2, using RO-3306, to allow for a clear distinction of the two centrosomes present at this cell cycle stage. Panels illustrate sum projections of the stacks from an original picture (left), a whole cell mask (middle) and a centrosome mask (right); both masks are based on intensity thresholding. Bar: 10 μm.


B ) EGFP - tagged viral like particles ( GFP - VLP2 / 6 ) are used as a reference for quantification of γ - tubulin - EGFP . The histogram shows the distribution of the total fluorescence intensity associated with single GFP - VLP2 / 6 ; the 2 dashed lines delimit the population used for assigning an average fluorescence intensity value to represent the 120 GFP molecules associated with each VLP2 / 6 particle ( Charpilienne et al , 2001 ) . The inset shows a sum projection of EGFP - VLPs fluorescence image . Bar : 10 μm .

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

B) EGFP-tagged viral like particles (GFP-VLP2/6) are used as a reference for quantification of γ-tubulin-EGFP. The histogram shows the distribution of the total fluorescence intensity associated with single GFP-VLP2/6; the 2 dashed lines delimit the population used for assigning an average fluorescence intensity value to represent the 120 GFP molecules associated with each VLP2/6 particle (Charpilienne et al, 2001). The inset shows a sum projection of EGFP-VLPs fluorescence image. Bar: 10 μm.


C ) Scatter plots show the numbers of γ - tubulin molecules per centrosome determined by calibrations using either SRM measurements ( n = 46 cells ) or EGFP - VLP2 / 6 fluorescence ( n = 40 cells ) . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scatter plots show the numbers of γ-tubulin molecules per centrosome determined by calibrations using either SRM measurements (n=46 cells) or EGFP-VLP2/6 fluorescence (n=40 cells). The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot.


To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification , endogenously tagged RPE - 1 cells were synchronized as described under Materials and Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 cells expressing ( endogenous ) γ - tubulin - EGFP ( A ) at the indicted cell cycle stages . Fluorescence images of representative cells are shown as maximum projections . Bars : 10μm . Bottom panels : Fluorescence quantifications were performed using the EGFP - VLP2 / 6 method for calibration . Scatter plots represent the number of γ - tubulin ( A ) molecules per centrosome at the indicated cell cycle stages . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot . The following numbers of cells were analyzed in early S phase : γ - tubulin : 45 ; in late S / G2 phase : γ - tubulin : 45 ; in late G2 phase : γ - tubulin : 48 ; in early mitosis : γ - tubulin : 31 . Note that fluorescence images were recorded using identical settings . P values from t - tests are indicated by the following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) and ns ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing (endogenous) γ-tubulin-EGFP (A) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of γ-tubulin (A) molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: γ-tubulin: 45; in late S/G2 phase: γ-tubulin: 45; in late G2 phase: γ-tubulin: 48; in early mitosis: γ-tubulin: 31. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).


To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification , endogenously tagged RPE - 1 cells were synchronized as described under Materials and Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 cells expressing Sas - 6 ( B ) at the indicted cell cycle stages . Fluorescence images of representative cells are shown as maximum projections . Bars : 10μm . Bottom panels : Fluorescence quantifications were performed using the EGFP - VLP2 / 6 method for calibration . Scatter plots represent the number of molecules per centrosome at the indicated cell cycle stages . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot . The following numbers of cells were analyzed in early S phase : Sas - 6 : 43 ; in late S / G2 phase : Sas - 6 : 46 ; in late G2 phase : Sas - 6 : 48 . Note that fluorescence images were recorded using identical settings . P values from t - tests are indicated by the following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) and ns ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing Sas-6 (B) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: Sas-6: 43; in late S/G2 phase: Sas-6: 46; in late G2 phase: Sas-6: 48. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).


To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification , endogenously tagged RPE - 1 cells were synchronized as described under Materials and Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 cells expressing STIL ( C ) at the indicted cell cycle stages . Fluorescence images of representative cells are shown as maximum projections . Bars : 10μm . Bottom panels : Fluorescence quantifications were performed using the EGFP - VLP2 / 6 method for calibration . Scatter plots represent the number of STIL ( C ) molecules per centrosome at the indicated cell cycle stages . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot . The following numbers of cells were analyzed in early S phase : STIL : 79 ; in late S / G2 phase : STIL : 68 ; in late G2 phase : STIL : 68 . Note that fluorescence images were recorded using identical settings . P values from t - tests are indicated by the following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) and ns ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing STIL (C) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of STIL (C) molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: STIL: 79; in late S/G2 phase: STIL: 68; in late G2 phase: STIL: 68. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).


a , Correlative light and electron microscopy of HeLa / GFP - LC3 cell infected with SIN / mCherry . capsid virus . Top left , deconvolved image of red and green fluorescence channels ; middle , three - dimensional surface reconstruction of red and green channels ; right , yellow ( red + green ) colocalization channel . Arrows denote yellow puncta that correspond to ' 1 ' and ' 2 ' in electron microscopy images below . Bottom left , electron microscopy of identical cell ; middle and right , high magnification images of insets ' 1 ' and ' 2 ' . Scale bars , left , 10 µm ; middle and right , 200 nm .

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a, Correlative light and electron microscopy of HeLa/GFP-LC3 cell infected with SIN/mCherry.capsid virus. Top left, deconvolved image of red and green fluorescence channels; middle, three-dimensional surface reconstruction of red and green channels; right, yellow (red + green) colocalization channel. Arrows denote yellow puncta that correspond to '1' and '2' in electron microscopy images below. Bottom left, electron microscopy of identical cell; middle and right, high magnification images of insets '1' and '2'. Scale bars, left, 10 µm; middle and right, 200 nm.


a , Electron microscopy analysis of wild - type ( WT ) and Smurf1 - / - ( KO ) MEFs grown in normal media or EBSS ( starvation ) for 4 h . Arrowheads , representative autophagosomes ; arrows , representative autolysosomes . Scale bars , 500 nm .

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a, Electron microscopy analysis of wild-type (WT) and Smurf1-/- (KO) MEFs grown in normal media or EBSS (starvation) for 4 h. Arrowheads, representative autophagosomes; arrows, representative autolysosomes. Scale bars, 500 nm.


b , Western blot analyses of LC3 - I / II ( non - lipidated and lipidated forms of MAP1LC3 , respectively ) and p62 levels in MEFs of indicated genotype .

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b, Western blot analyses of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in MEFs of indicated genotype.


c , Quantification of colocalization of SIN / mCherry . capsid and GFP - LC3 in indicated MEFs 16 h after SIN / mCherry . capsid / GFP - LC3 infection . Data shown represent mean ± s . e . m . number of colocalized red and green puncta per cell for 50 cells per well in triplicate samples . * P < 0 . 001 against wild - type , Students t - test .

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c, Quantification of colocalization of SIN/mCherry.capsid and GFP-LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP-LC3 infection. Data shown represent mean ± s.e.m. number of colocalized red and green puncta per cell for 50 cells per well in triplicate samples. *P<0.001 against wild-type, Students t-test.


d , Representative confocal micrographs of colocalization of SIN / mCherry . capsid and GFP - LC3 in indicated MEFs 16 h after SIN / mCherry . capsid / GFP - LC3 infection images used for quantification in c . Arrows , colocalized red and green puncta . Scale bar , 15 & amp ; amp ; mgr ; m .

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d, Representative confocal micrographs of colocalization of SIN/mCherry.capsid and GFP-LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP-LC3 infection images used for quantification in c. Arrows, colocalized red and green puncta. Scale bar, 15 &amp;mgr;m.


e , Representative electron microscopy images of indicated MEFs infected with HSV - 1 ( strain 17termA ) . White arrowheads , partially degraded viral nucleocapsids inside autolysosomes ; black arrowheads , intact viral nucleocapsids inside viral vesicles . Scale bar , 0 . 5 µm . For a - e , similar results were obtained in 3 - 5 independent experiments .

e, Representative electron microscopy images of indicated MEFs infected with HSV-1 (strain 17termA). White arrowheads, partially degraded viral nucleocapsids inside autolysosomes; black arrowheads, intact viral nucleocapsids inside viral vesicles. Scale bar, 0.5 µm. For a-e, similar results were obtained in 3-5 independent experiments.


a , Representative mitochondrial morphology in Smurf1 + / + ( wild - type ) and Smurf1 − / − ( KO ) MEFs transfected with indicated construct and treated with DMSO or 10 µM CCCP for 24 h . b , Quantification of percentage of total cells with a diffuse accumulation of abnormal fragmented mitochondria and lack of mitochondrial clearance . Results shown represent combined data from 3 - 5 experiments per condition with triplicate wells ( of at least 100 cells per well ) analysed for each condition per experiment . Shown are mean ± s . e . m . for average values from each experiment . Similar results were observed in each independent experiment . * P 0 . 001 , Students t - test . c , Measurement of mitochondrial fractional area ( percentage of total cellular area ) in MEFs treated as in a . Results shown represent mean ± s . e . m . for 50 cells per condition .

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a, Representative mitochondrial morphology in Smurf1+/+ (wild-type) and Smurf1−/− (KO) MEFs transfected with indicated construct and treated with DMSO or 10 µM CCCP for 24 h. b, Quantification of percentage of total cells with a diffuse accumulation of abnormal fragmented mitochondria and lack of mitochondrial clearance. Results shown represent combined data from 3-5 experiments per condition with triplicate wells (of at least 100 cells per well) analysed for each condition per experiment. Shown are mean ± s.e.m. for average values from each experiment. Similar results were observed in each independent experiment. *P  0.001, Students t-test. c, Measurement of mitochondrial fractional area (percentage of total cellular area) in MEFs treated as in a. Results shown represent mean ± s.e.m. for 50 cells per condition.


d , Representative confocal micrographs of KO MEFs transfected with YFP - SMURF1 wild - type or YFP - SMURF1Dgr ; C2 ( Dgr ; C2 ) and treated for 4 h with DMSO or CCCP .

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d, Representative confocal micrographs of KO MEFs transfected with YFP-SMURF1 wild-type or YFP-SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with DMSO or CCCP.


e , Representative confocal micrographs of KO MEFs transfected with GFP - LC3 and wild - type mCherry - SMURF1 ( WT ) or mCherry - SMURF1Dgr ; C2 ( Dgr ; C2 ) and treated for 4 h with CCCP . Inset , upper right , formation of completed autophagosome around a damaged mitochondrion associated with wild - type SMURF1 ; insets , lower right , incomplete autophagosomes or absence of LC3 signal around mitochondria associated with SMURF1Dgr ; C2 . See also Supplementary Figs 10 and 11 for enlarged images .

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e, Representative confocal micrographs of KO MEFs transfected with GFP-LC3 and wild-type mCherry-SMURF1 (WT) or mCherry-SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with CCCP. Inset, upper right, formation of completed autophagosome around a damaged mitochondrion associated with wild-type SMURF1; insets, lower right, incomplete autophagosomes or absence of LC3 signal around mitochondria associated with SMURF1Dgr;C2. See also Supplementary Figs 10 and 11 for enlarged images.


f , Representative electron microscopy images of indicated tissues from 10 - month - oldSmurf1 + / + ( WT ) or Smurf1 - / - ( KO ) mice . Arrows , representative abnormal mitochondria . Scale bars , 1 µm . Similar abnormalities were observed throughout entire electron microscopy tissue section for each mouse and in tissue samples from three different mice for each genotype .

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f, Representative electron microscopy images of indicated tissues from 10-month-oldSmurf1+/+ (WT) or Smurf1-/- (KO) mice. Arrows, representative abnormal mitochondria. Scale bars, 1 µm. Similar abnormalities were observed throughout entire electron microscopy tissue section for each mouse and in tissue samples from three different mice for each genotype.


B Latency to enter dark compartment for Gadd45a - WT ( white bars , n = 11 ) and Gadd45a - KO ( blue bars , n = 11 ) . A significant reduction was found in the Gadd45a - KO at 24 hours and 7 days after the training . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni post - hoc test : * = p < 0 . 05 .

B Latency to enter dark compartment for Gadd45a-WT (white bars, n=11) and Gadd45a-KO (blue bars, n=11). A significant reduction was found in the Gadd45a-KO at 24 hours and 7 days after the training. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05.


C In situ hybridization to detect Gadd45a mRNA in adult wild - type hippocampus ( n = 8 animals ; antisense , left panel ; sense , middle panel ) and in Gadd45a - KO sections ( n = 6 animals ; right panel ) . Scale bars : 500 µm ( black bar ) .

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C In situ hybridization to detect Gadd45a mRNA in adult wild-type hippocampus (n=8 animals; antisense, left panel; sense, middle panel) and in Gadd45a-KO sections (n=6 animals; right panel). Scale bars: 500 µm (black bar).


D Experience - dependent changes in mRNA levels were evaluated under control conditions ( home cage ) and exposed to the PA ( context , and context + shock groups ; n = 6 for all groups ) , respectively . Hippocampal Gadd45a mRNA levels remained unchanged , while Gadd45b was induced ( context ; t10 = 2 . 427 , context + shock t10 = 2 . 319 ) . Concomitantly , mRNA levels of activity - regulated cytoskeleton protein ( Arc ) , as a proxy of neuronal activation , were increased in mice exposed to the PA context ( t10 = 3 . 406 ) and context + shock ( t10 = 3 . 141 ) . Values shown are mean ± SEM ; 1 - Way ANOVA and unpaired t - test : * = p < 0 . 05 , * * * = p < 0 . 001 .

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D Experience-dependent changes in mRNA levels were evaluated under control conditions (home cage) and exposed to the PA (context, and context + shock groups; n=6 for all groups), respectively. Hippocampal Gadd45a mRNA levels remained unchanged, while Gadd45b was induced (context; t10=2.427, context + shock t10=2.319). Concomitantly, mRNA levels of activity-regulated cytoskeleton protein (Arc), as a proxy of neuronal activation, were increased in mice exposed to the PA context (t10=3.406) and context + shock (t10=3.141). Values shown are mean ± SEM; 1-Way ANOVA and unpaired t-test: * = p<0.05, *** = p<0.001.


Gadd45a mRNA level in the hippocampus of Gadd45a - overexpressing mice ( red , n = 7 ) was significantly increased ( t10 = 4 . 189 ) as compared to Gadd45a - WT ( n = 5 ) . Values shown are mean ± SEM ; unpaired t - test : * * = p < 0 . 01 .

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Gadd45a mRNA level in the hippocampus of Gadd45a-overexpressing mice (red, n=7) was significantly increased (t10=4.189) as compared to Gadd45a-WT (n=5). Values shown are mean ± SEM; unpaired t-test: ** = p<0.01.


C Immunohistochemistry for hemagglutinin ( HA , fused to Gadd45α ) in Gadd45a - WT ( Nex - Cre ( - / - ) , n = 5 ) and Gadd45a - overexpressing hippocampi ( Nex - Cre ( + / - ) , n = 5 ) 8 weeks after AAV injection . Note that the Gadd45α - HA signal in Gadd45a - overexpressing mice is predominantly found in the stratum pyramidale ( sp ) but also in the stratum oriens and stratum radiatum ( so and sr , respectively ) . Scale bar : 250 µm .

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C Immunohistochemistry for hemagglutinin (HA, fused to Gadd45α) in Gadd45a-WT (Nex-Cre(-/-), n=5) and Gadd45a-overexpressing hippocampi (Nex-Cre(+/-), n=5) 8 weeks after AAV injection. Note that the Gadd45α-HA signal in Gadd45a-overexpressing mice is predominantly found in the stratum pyramidale (sp) but also in the stratum oriens and stratum radiatum (so and sr, respectively). Scale bar: 250 µm.


D Cellular distribution of Gadd45α was analyzed by HA - western blot on nuclear and cytoplasmic protein fractions of Gadd45a - WT and Gadd45a - overexpressing mice ( n = 2 for both groups ) . HA was found predominantly in the nuclear fraction of Gadd45a - overexpressing hippocampi , with a smaller but recognizable expression also in the cytoplasm .

D Cellular distribution of Gadd45α was analyzed by HA-western blot on nuclear and cytoplasmic protein fractions of Gadd45a-WT and Gadd45a-overexpressing mice (n=2 for both groups). HA was found predominantly in the nuclear fraction of Gadd45a-overexpressing hippocampi, with a smaller but recognizable expression also in the cytoplasm.


E Latency to enter the dark compartment for Gadd45a - WT ( white bars , n = 10 ) and Gadd45a - overexpressing mice ( red bars , n = 12 ) in the PA test . A significant increase was found 24 hours and 7 days after the PA training in the Gadd45a - overexpresion group . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni Post - hoc test : * = 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 O O O O O O O O O O O O O O O O O O O O O O O

E Latency to enter the dark compartment for Gadd45a-WT (white bars, n=10) and Gadd45a-overexpressing mice (red bars, n=12) in the PA test. A significant increase was found 24 hours and 7 days after the PA training in the Gadd45a-overexpresion group. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: * = p<0.05.


B LTP protocol was applied to 16 slices of Gadd45a - WT mice ( white dots , n = 5 animals ) and 11 slices of Gadd45a - KO mice ( blue dots , n = 5 animals ) , resulting in a significantly ( p < 0 . 05 , Student t - test ) lowered induction of LTP in Gadd45a - KO mice as compared with Gadd45a - WT . Values shown are mean ± SEM . C Gadd45a - overexpressing mice ( red dots , n = 10 slices from 6 animals ) presented a slightly increased LTP as compared with Gadd45a - WT ( white dots , n = 8 slices from 6 animals ) , which became more pronounced during the late phase of the experiment . Values shown are mean ± SEM .

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B LTP protocol was applied to 16 slices of Gadd45a-WT mice (white dots, n=5 animals) and 11 slices of Gadd45a-KO mice (blue dots, n=5 animals), resulting in a significantly (p<0.05, Student t-test) lowered induction of LTP in Gadd45a-KO mice as compared with Gadd45a-WT. Values shown are mean ± SEM. C Gadd45a-overexpressing mice (red dots, n=10 slices from 6 animals) presented a slightly increased LTP as compared with Gadd45a-WT (white dots, n=8 slices from 6 animals), which became more pronounced during the late phase of the experiment. Values shown are mean ± SEM.


A MA plot of DESeq differential expression analysis with the 1 % false discovery rate ( FDR ) hits in red ( 5 / 402 ; and with > 2 - fold change 1 / 65 , up - / down - regulated respectively ) .

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A MA plot of DESeq differential expression analysis with the 1% false discovery rate (FDR) hits in red (5/402; and with >2-fold change 1/65, up-/down-regulated respectively).


D UCSC browser view of reads - per - million - normalised RNA - seq coverage for Grin2a in Gadd45a - WT ( black ) and Gadd45a - KO ( blue ) representative samples . Gene structure of Grin2a ( colored in purple ) , and the direction of transcription ( red arrow ) . Upper pannel covers the entire transcript while lower panel is an enlargement of exons 4 - 8 .

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D UCSC browser view of reads-per-million-normalised RNA-seq coverage for Grin2a in Gadd45a-WT (black) and Gadd45a-KO (blue) representative samples. Gene structure of Grin2a (colored in purple), and the direction of transcription (red arrow). Upper pannel covers the entire transcript while lower panel is an enlargement of exons 4-8.


E Detailed view of the extended 3 ´ UTR of Grin2a showing a significant 5 ´ < 3 ´ gradient in RNA - seq coverage . Note that the normalised read coverage is similar between genotypes at the actual 3 ´ UTR end but differs significantly upstream including the annotated exons . For subsequent experiments , position of the designed primers covering proximal and distal parts of the 3 ´ UTR are indicated below .

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E Detailed view of the extended 3´UTR of Grin2a showing a significant 5´<3´ gradient in RNA-seq coverage. Note that the normalised read coverage is similar between genotypes at the actual 3´UTR end but differs significantly upstream including the annotated exons. For subsequent experiments, position of the designed primers covering proximal and distal parts of the 3´UTR are indicated below.


F , G Validation of Gadd45α - regulated levels of Grin2a mRNA by qPCR with primers covering exons 3 - 4 . Grin2a mRNA levels were analyzed in the hippocampus of mice under control conditions ( white bars ; Gadd45a - WT = 7 and Gadd45a - KO = 7 ) and 1 hour after PA ( Gadd45a - WT = 5 , black bars ; Gadd45a - KO = 6 , blue bars ) . Random primed cDNA levels ( F ) , were very similar in all the experimental groups . Oligo ( dT ) - primed cDNA ( G ) revealed a significant increase of Grin2a 1h after PA , which was only observed in Gadd45a - WT mice . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni post - hoc test : * * = p < 0 . 01 , * * * = p < 0 . 001 . H , I mRNA levels of proximal vs . distal parts of the 3 ´ UTR analyzed in the same set of oligo ( dT ) - primed cDNA . ( H ) Significant increase in Grin2a mRNA levels was found for the proximal part only in Gadd45a - WT . ( I ) Distal parts of the 3 ´ UTR remained unchanged ( n = 6 for all groups ) . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni post - hoc test : * = p < 0 . 05 .

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F, G Validation of Gadd45α-regulated levels of Grin2a mRNA by qPCR with primers covering exons 3-4. Grin2a mRNA levels were analyzed in the hippocampus of mice under control conditions (white bars; Gadd45a-WT=7 and Gadd45a-KO=7) and 1 hour after PA (Gadd45a-WT=5, black bars; Gadd45a-KO=6, blue bars). Random primed cDNA levels (F), were very similar in all the experimental groups. Oligo(dT)-primed cDNA (G) revealed a significant increase of Grin2a 1h after PA, which was only observed in Gadd45a-WT mice. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: ** = p<0.01, *** = p<0.001. H, I mRNA levels of proximal vs. distal parts of the 3´UTR analyzed in the same set of oligo(dT)-primed cDNA. (H) Significant increase in Grin2a mRNA levels was found for the proximal part only in Gadd45a-WT. (I) Distal parts of the 3´UTR remained unchanged (n=6 for all groups). Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05.


A , B NR2A protein levels were analyzed in the hippocampus of mice under control conditions ( white bars ; Gadd45a - WT , n = 13 and Gadd45a - KO , n = 14 ) and 1 hour after PA ( black bars ; Gadd45a - WT = 14 , Gadd45a - KO = 14 ) . NR2A signal ( 160 kDa ) was normalized to the loading control ( tubulin ( 55 kDa ) for the total fraction , and synaptotagmin ( 68 kDa ) for the synaptosomal fraction ) and calculated as percentage of control . In Gadd45a - WT samples ( A ) , NR2A protein levels were unchanged in the total fraction during memory formation , while in the synaptosomal fraction , a significant increase was detected ( t20 = 3 . 199 ) . In Gadd45a - KO hippocampi ( B ) , levels of NR2A protein in both fractions remained unaltered during memory consolidation . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni post - hoc test : * * = p < 0 . 01 .

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A, B NR2A protein levels were analyzed in the hippocampus of mice under control conditions (white bars; Gadd45a-WT, n=13 and Gadd45a-KO, n=14) and 1 hour after PA (black bars; Gadd45a-WT=14, Gadd45a-KO=14). NR2A signal (160 kDa) was normalized to the loading control (tubulin (55 kDa) for the total fraction, and synaptotagmin (68 kDa) for the synaptosomal fraction) and calculated as percentage of control. In Gadd45a-WT samples (A), NR2A protein levels were unchanged in the total fraction during memory formation, while in the synaptosomal fraction, a significant increase was detected (t20=3.199). In Gadd45a-KO hippocampi (B), levels of NR2A protein in both fractions remained unaltered during memory consolidation. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: ** = p<0.01.


C Gadd45α pulldown experiments were carried out in wild - type hippocampal lysates ( n = 12 for all groups ) . Percentage of input recovery was unchanged between GFP - FLAG ( green bars ) and Gadd45α - FLAG ( black bars ) for genes that are not regulated by Gadd45α , such as succinate dehydrogenase complex flavoprotein subunit A ( Sdha , no 3 ´ UTR extension ) , and mitogen - activated protein kinase 6 ( Map2K6 , containing 3 ´ UTR extension of 10 kb ) . On the contrary , Gadd45α - FLAG bound fraction revealed a significant increase in Grin2a ( t22 = 4 . 992 ) and Grm5 mRNA ( t22 = 4 . 939 ) as compared to GFP - FLAG fraction . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni Post - hoc test : * * * = p < 0 . 001 .

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C Gadd45α pulldown experiments were carried out in wild-type hippocampal lysates (n=12 for all groups). Percentage of input recovery was unchanged between GFP-FLAG (green bars) and Gadd45α-FLAG (black bars) for genes that are not regulated by Gadd45α, such as succinate dehydrogenase complex flavoprotein subunit A (Sdha, no 3´UTR extension), and mitogen-activated protein kinase 6 (Map2K6, containing 3´UTR extension of 10 kb). On the contrary, Gadd45α-FLAG bound fraction revealed a significant increase in Grin2a (t22=4.992) and Grm5 mRNA (t22=4.939) as compared to GFP-FLAG fraction. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: *** = p<0.001.


B Hippocampal Gadd45a mRNA levels were significantly upregulated in both Gadd45a - overexpression ( q24 = 4 . 094 ) and Gadd45aK45E - overexpression groups ( q24 = 4 . 726 ) ( n = 9 for all groups ) . Values shown are mean ± SEM ; Tukey ´ s multiple comparison test : * = p < 0 . 05 , * * = p < 0 . 01 .

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B Hippocampal Gadd45a mRNA levels were significantly upregulated in both Gadd45a-overexpression (q24 = 4.094) and Gadd45aK45E-overexpression groups (q24 = 4.726) (n=9 for all groups). Values shown are mean ± SEM; Tukey´s multiple comparison test: * = p<0.05, ** = p<0.01.


C Immunohistochemistry for hemagglutinin ( HA , fused to Gadd45α ) in Gadd45a - WT ( left ) , Gadd45a - overexpression ( middle ) and Gadd45aK45E - overexpression ( right ) samples 8 weeks after AAV injection ( n = 3 for all groups ) . Upper row depicts the entire hippocampus , while center and lower rows show magnifications of the CA3 area in which the HA signal ( red ) is not restricted to the nuclei ( blue and white ) . Scale bar : 250 µm .

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C Immunohistochemistry for hemagglutinin (HA, fused to Gadd45α) in Gadd45a-WT (left), Gadd45a-overexpression (middle) and Gadd45aK45E-overexpression (right) samples 8 weeks after AAV injection (n=3 for all groups). Upper row depicts the entire hippocampus, while center and lower rows show magnifications of the CA3 area in which the HA signal (red) is not restricted to the nuclei (blue and white). Scale bar: 250 µm.


D Gadd45α - overexpression was confirmed at the protein level by HA - western blot on hippocampal samples from Gadd45a - WT , Gadd45a - overexpression and Gadd45aK45E - overexpression mice ( n = 3 for all groups ) . Hippocampus scale bars : 250 µm ; CA3 scale bars : 50µm .

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D Gadd45α-overexpression was confirmed at the protein level by HA-western blot on hippocampal samples from Gadd45a-WT, Gadd45a-overexpression and Gadd45aK45E-overexpression mice (n=3 for all groups). Hippocampus scale bars: 250 µm; CA3 scale bars: 50µm.


E Latency to enter the dark compartment for Gadd45a - WT ( white bars , n = 12 ) , Gadd45a - overexpression ( red bars , n = 11 ) and Gadd45aK45E - overexpression mice ( grey bars , n = 11 ) in the PA test . Note that , unlike Gadd45a - overexpression mice , those expressing the mutated form of Gadd45α ( no RNA - binding capacity ) did not show an increase in memory retention . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni Post - hoc test : * = p < 0 . 05 , * * * = p < 0 . 001 .

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E Latency to enter the dark compartment for Gadd45a-WT (white bars, n=12), Gadd45a-overexpression (red bars, n=11) and Gadd45aK45E-overexpression mice (grey bars, n=11) in the PA test. Note that, unlike Gadd45a-overexpression mice, those expressing the mutated form of Gadd45α (no RNA-binding capacity) did not show an increase in memory retention. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: * = p<0.05, *** = p<0.001.


A . Mutations in the kinase motif abolished the yeast toxicity of MavQ . S . cerevisiae was transformed with plasmids expressing Flag - tagged wild - type MavQ or the indicated MavQ mutants under the galactose - inducible promoter . Yeast cells were spotted on synthetic medium supplemented with glucose or galactose for 3 days before image acquisition .

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A. Mutations in the kinase motif abolished the yeast toxicity of MavQ. S. cerevisiae was transformed with plasmids expressing Flag-tagged wild-type MavQ or the indicated MavQ mutants under the galactose-inducible promoter. Yeast cells were spotted on synthetic medium supplemented with glucose or galactose for 3 days before image acquisition.


B . The expression of MavQ in the yeast was detected by Flag antibody . The PGK ( 3 - phosphoglyceric phosphokinase ) was used as a loading control .

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B. The expression of MavQ in the yeast was detected by Flag antibody. The PGK (3-phosphoglyceric phosphokinase) was used as a loading control.


A . Biochemical assays for the kinase activity of MavQ . Purified MavQ was incubated with or without specific PI substrates , the conversion of ATP to ADP was measured by the ADP - Glo assay . RLU , relative luminescence units .

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A. Biochemical assays for the kinase activity of MavQ. Purified MavQ was incubated with or without specific PI substrates, the conversion of ATP to ADP was measured by the ADP-Glo assay. RLU, relative luminescence units.


B . H149 , N152 and D160 in MavQ are critical for its kinase activity . Purified wild - type and mutant MavQ were reacted with PtdIns and ATP . The kinase activity was determined by the ADP - Glo assay .

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B. H149, N152 and D160 in MavQ are critical for its kinase activity. Purified wild-type and mutant MavQ were reacted with PtdIns and ATP. The kinase activity was determined by the ADP-Glo assay.


C . The PI product produced by MavQ determined by TLC assays . di - C8 - Bodipy - FL - PtdIns was used to react with MavQ ( lane 4 ) and samples were further incubated with the 3 - phosphatase MTM which hydrolyzes both PtdIns3P and PtdIns ( 3 , 5 ) P2 ( lane 5 ) or with the PI4K LepB _ NTD which phosphorylates PtdIns3P into PtdIns ( 3 , 4 ) P2 ( lane 6 ) . A schematic illustration of the enzymatic reactions is shown in the lower panel .

O 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 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 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 B-MEASURED_VAR 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

C. The PI product produced by MavQ determined by TLC assays. di-C8-Bodipy-FL-PtdIns was used to react with MavQ (lane 4) and samples were further incubated with the 3-phosphatase MTM which hydrolyzes both PtdIns3P and PtdIns(3,5)P2 (lane 5) or with the PI4K LepB_NTD which phosphorylates PtdIns3P into PtdIns (3,4)P2 (lane 6). A schematic illustration of the enzymatic reactions is shown in the lower panel.


D . Mutations in H149 , N152 and D160 abolish the ability of MavQ to convert PtdIns into PtdIns3P .

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

D. Mutations in H149, N152 and D160 abolish the ability of MavQ to convert PtdIns into PtdIns3P.


A . Representative fluorescence images of HeLa cells expressing the PtdIns3P probe GFP - 2xFYVEHrs and MavQ with or without wortmannin treatment . Scale bars , 5 μm .

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A. Representative fluorescence images of HeLa cells expressing the PtdIns3P probe GFP-2xFYVEHrs and MavQ with or without wortmannin treatment. Scale bars, 5 μm.


B . Percentage of HeLa cells transfected with the indicated plasmids in which GFP - 2xFYVEHrs localizes to vesicle - like structures . At least 100 cells ( n ≥ 100 ) were counted for each sample . Wort , wortmannin .

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B. Percentage of HeLa cells transfected with the indicated plasmids in which GFP-2xFYVEHrs localizes to vesicle-like structures. At least 100 cells (n≥100) were counted for each sample. Wort, wortmannin.


C . Induction of the ATP hydrolysis activity of MavQ by PtdIns is not affected by wortmannin . Recombinant MavQ was incubated with increasing amounts of wortmannin for 30 min at room temperature prior to the addition of PtdIns and ATP . The wortamannin - sensitive PI3K p110α was used as a control . The ADP - Glo assay was used to measure the kinase activity .

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C. Induction of the ATP hydrolysis activity of MavQ by PtdIns is not affected by wortmannin. Recombinant MavQ was incubated with increasing amounts of wortmannin for 30 min at room temperature prior to the addition of PtdIns and ATP. The wortamannin-sensitive PI3K p110α was used as a control. The ADP-Glo assay was used to measure the kinase activity.


D . Wortmannin does not affect the production of PtdIns3P by MavQ . Purified MavQ was pretreated with 100 μM of wortmannin for 30 min at room temperature . di - C8 - Bodipy - FL - PtdIns and ATP were then added to the reaction mixtures . The TLC assay was employed to detect the generation of PtdIns3P by MavQ . The wortamannin - sensitive PI3K p110α was included as a control .

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D. Wortmannin does not affect the production of PtdIns3P by MavQ. Purified MavQ was pretreated with 100 μM of wortmannin for 30 min at room temperature. di-C8-Bodipy-FL-PtdIns and ATP were then added to the reaction mixtures. The TLC assay was employed to detect the generation of PtdIns3P by MavQ. The wortamannin-sensitive PI3K p110α was included as a control.


A . Representative immunofluorescence images of the association of MavQ with the LCV . BMDM cells infected with wild - type or Dot / Icm - deficient ( dotA - ) L . pneumophila were sequentially stained with antibodies against L . pneumophila and MavQ . Images were taken under a fluorescence microscope . Insets represent 3x magnification of regions defined by dash lines . Scale bar , 5 μm .

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A. Representative immunofluorescence images of the association of MavQ with the LCV. BMDM cells infected with wild-type or Dot/Icm-deficient (dotA-) L. pneumophila were sequentially stained with antibodies against L. pneumophila and MavQ. Images were taken under a fluorescence microscope. Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.


B . The rates of of LCVs positively stained for MavQ 2 h post - infection . At least 100 LCVs ( n ≥ 100 ) were scored for each sample .

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B. The rates of of LCVs positively stained for MavQ 2 h post-infection. At least 100 LCVs (n≥100) were scored for each sample.


C . Dynamics of MavQ association with LCV . BMDMs were infected with the wild - type L . pneumophila strain at an MOI of 2 for the indicated experimental durations . At least 100 phagosomes ( n ≥ 100 ) were counted for each sample to obtain the percentage of MavQ - positive LCVs .

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C. Dynamics of MavQ association with LCV. BMDMs were infected with the wild-type L. pneumophila strain at an MOI of 2 for the indicated experimental durations. At least 100 phagosomes (n≥100) were counted for each sample to obtain the percentage of MavQ-positive LCVs.


D . Intracellular growth of L . pneumophila in macrophages . BMDMs were infected with indicated L . pneumophila strains at an MOI of 0 . 05 . At the indicated time points , CFUs were determined by plating the saponin - solubilized lysates of infected cells on CYE plates .

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D. Intracellular growth of L. pneumophila in macrophages. BMDMs were infected with indicated L. pneumophila strains at an MOI of 0.05. At the indicated time points, CFUs were determined by plating the saponin-solubilized lysates of infected cells on CYE plates.


E . Expression of mavQ in L . pneumophila strains used for infection . Total proteins of bacteria cultured to the post - exponential phase separated by SDS - PAGE were probed with MavQ - specific antibodies . ICDH was detected as a loading control .

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E. Expression of mavQ in L. pneumophila strains used for infection. Total proteins of bacteria cultured to the post-exponential phase separated by SDS-PAGE were probed with MavQ-specific antibodies. ICDH was detected as a loading control.


A . Biochemical synthesis of PtdIns4P from PtdIns by sequential reactions catalyzed by MavQ , LepB _ NTD and SidF . MavQ was reacted with di - C8 - Bodipy - FL - PtdIns , resulting in the production of PtdIns3P ( lane 6 ) . The PtdIns3P - specific PI4K LepB - NTD was then added into the reaction , yielding PtdIns ( 3 , 4 ) P2 ( lane 7 ) . The product of the sample receiving LepB _ NTD was further incubated with the 3 - phosphatase SidF to yield PtdIns4P ( lane 8 ) . The lipid products were detected and visualized by TLC . A schematic illustration of the enzymatic reactions is shown in the lower panel .

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A. Biochemical synthesis of PtdIns4P from PtdIns by sequential reactions catalyzed by MavQ, LepB_NTD and SidF. MavQ was reacted with di-C8-Bodipy-FL-PtdIns, resulting in the production of PtdIns3P (lane 6). The PtdIns3P-specific PI4K LepB-NTD was then added into the reaction, yielding PtdIns(3,4)P2 (lane 7). The product of the sample receiving LepB_NTD was further incubated with the 3-phosphatase SidF to yield PtdIns4P (lane 8). The lipid products were detected and visualized by TLC. A schematic illustration of the enzymatic reactions is shown in the lower panel.


B . Representative images of SidC anchoring on LCVs . BMDMs infected with the indicated L . pneumophila strains at an MOI of 2 for 2 h were sequentially stained with antibodies against the bacteria and SidC . Images were acquired using a fluorescence microscope . Insets represent 3x magnification of regions defined by dash lines . Scale bar , 5 μm .

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B. Representative images of SidC anchoring on LCVs. BMDMs infected with the indicated L. pneumophila strains at an MOI of 2 for 2 h were sequentially stained with antibodies against the bacteria and SidC. Images were acquired using a fluorescence microscope. Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.


C . Quantitation of SidC - positive LCVs . At least 100 phagosomes ( n ≥ 100 ) were scored for each sample .

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C. Quantitation of SidC-positive LCVs. At least 100 phagosomes (n≥100) were scored for each sample.


D . Expression and translocation of SidC . The lysates of L . pneumophila strains used for infection were probed for protein expression , ICDH was detected as a loading control ( top panel ) . SidC translocation was detected by probing the saponin - soluble fractions of infected U937 cells with SidC - specific antibodies . Tubulin was probed as a loading control ( bottom panel ) .

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D. Expression and translocation of SidC. The lysates of L. pneumophila strains used for infection were probed for protein expression, ICDH was detected as a loading control (top panel). SidC translocation was detected by probing the saponin-soluble fractions of infected U937 cells with SidC-specific antibodies. Tubulin was probed as a loading control (bottom panel).


A . Direct interactions between SidP and MavQ in the GST pull - down assay . GST beads coated with GST - SidP or GST were incubated with His6 - MavQ . After washing the beads with GST binding buffer , the proteins resolved by SDS - PAGE were detected by Coomassie brilliant blue staining .

A. Direct interactions between SidP and MavQ in the GST pull-down assay. GST beads coated with GST-SidP or GST were incubated with His6-MavQ. After washing the beads with GST binding buffer, the proteins resolved by SDS-PAGE were detected by Coomassie brilliant blue staining.


B , SidP does not impair the kinase activity of MavQ in vitro . MavQ was preincubated with increasing amounts of SidP , and the kinase activity of MavQ was measured by the ADP - Glo assay ( B )

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B, SidP does not impair the kinase activity of MavQ in vitro. MavQ was preincubated with increasing amounts of SidP, and the kinase activity of MavQ was measured by the ADP-Glo assay (B)


C . SidP does not impair the kinase activity of MavQ in vitro . MavQ was preincubated with increasing amounts of SidP , and the kinase activity of MavQ was measured by TLC assay ( C ) .

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C. SidP does not impair the kinase activity of MavQ in vitro. MavQ was preincubated with increasing amounts of SidP, and the kinase activity of MavQ was measured by TLC assay (C).


D . Co - expression of SidP with MavQ in HeLa cells does not influence the production of PtdIns3P caused by MavQ . The association of the fluorescence probe GFP - 2xFYVEHrs with vesicle - like structures was used to indicate the distribution of PtdIns3P in cells . At least 100 cells were scored in each sample ( n ≥ 100 ) ( left panel ) . Wort , wortmannin . Expression of MavQ and SidP in the samples were shown in the right panel . EV represents empty vector .

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D. Co-expression of SidP with MavQ in HeLa cells does not influence the production of PtdIns3P caused by MavQ. The association of the fluorescence probe GFP-2xFYVEHrs with vesicle-like structures was used to indicate the distribution of PtdIns3P in cells. At least 100 cells were scored in each sample (n≥100) (left panel). Wort, wortmannin. Expression of MavQ and SidP in the samples were shown in the right panel. EV represents empty vector.


E . Deletion of sidP does not impact the association of SidC with the LCV . BMDMs infected with the indicated L . pneumophila stains were stained and the association of SidC with bacterial phagosomes was similarly determined ( n ≥ 100 ) ( left panel ) . Representative images of infected cells harboring bacterial phagosomes were also shown ( right panel ) . Insets represent 3x magnification of regions defined by dash lines . Scale bar , 5 μm .

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E. Deletion of sidP does not impact the association of SidC with the LCV. BMDMs infected with the indicated L. pneumophila stains were stained and the association of SidC with bacterial phagosomes was similarly determined (n≥100) (left panel). Representative images of infected cells harboring bacterial phagosomes were also shown (right panel). Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.


A WT MDFs were treated ± TSI individually or in combination for 3 hrs . Whole cell lysates ( WCL ) and UBA - pull down ( UBA - PD ) fractions were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments . Samples of UBA - pull down in following figures were analysed in the same way unless otherwise indicated .

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A WT MDFs were treated ± TSI individually or in combination for 3 hrs. Whole cell lysates (WCL) and UBA-pull down (UBA-PD) fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments. Samples of UBA-pull down in following figures were analysed in the same way unless otherwise indicated.


B WT , Ripk3 - / - , Mlkl - / - , Tnfr1 - / - and Traf2 - / - MDFs were untreated ( UT ) or treated with TSI for 3 hrs . Nec - 1 and GSK872 were added to inhibit RIPK1 and RIPK3 kinase activities respectively .

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B WT, Ripk3-/-, Mlkl-/-, Tnfr1-/- and Traf2-/- MDFs were untreated (UT) or treated with TSI for 3 hrs. Nec-1 and GSK872 were added to inhibit RIPK1 and RIPK3 kinase activities respectively.


E RIPK3 - gyrase were inducibly expressed in Ripk3 - / - MDFs by doxycycline ( dox ) for 5 hrs , and cells were then treated ± combination of TSI , or coumermycin ( coum ) for 3 hrs .

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E RIPK3-gyrase were inducibly expressed in Ripk3-/- MDFs by doxycycline (dox) for 5 hrs, and cells were then treated ± combination of TSI, or coumermycin (coum) for 3 hrs.


B UBA - pull down from WT MDFs treated with TSI for 3 hrs were subjected to the DUBs shown in ( A ) . Beads eluates were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments .

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B UBA-pull down from WT MDFs treated with TSI for 3 hrs were subjected to the DUBs shown in (A). Beads eluates were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.


C 1 . 4 mL cleared cell lysate from 4 x 106 TSI - treated WT MDFs was split into three parts of the indicated volume , followed by UBA - pull down and DUB incubation . Bead eluates were analysed by Western blot and probed with the indicated antibodies . Representative of three independent experiments .

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C 1.4 mL cleared cell lysate from 4 x 106 TSI-treated WT MDFs was split into three parts of the indicated volume, followed by UBA-pull down and DUB incubation. Bead eluates were analysed by Western blot and probed with the indicated antibodies. Representative of three independent experiments.


A WT MDFs were treated with TSI for indicated time . Cells were fractionated into cytosol and crude membrane , followed by UBA - pull down . All fractions were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments .

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A WT MDFs were treated with TSI for indicated time. Cells were fractionated into cytosol and crude membrane, followed by UBA-pull down. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.


B WT USP21 - CaaX and USP21C221R - CaaX ( CR ) were inducibly expressed in WT MDFs by doxycycline for 5 hrs . Ubiquitylated proteins were enriched followed by TSI stimulation and cellular fractionation . All fractions were analysed by Western blot and probed with antibodies as indicated . Representative of three independent experiments .

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B WT USP21-CaaX and USP21C221R-CaaX (CR) were inducibly expressed in WT MDFs by doxycycline for 5 hrs. Ubiquitylated proteins were enriched followed by TSI stimulation and cellular fractionation. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.


A WT and R105AD106A mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline , at the same time cells were untreated ( UT ) or treated with TSQ for 6 hrs . Cells were fractionated into cytosol ( C ) and crude membrane ( M ) . Fractions were analysed by BN - or SDS - PAGE , Western blot and probed with the indicated antibodies . Representative of three independent experiments . B Cell lysates from ( A ) were subjected to UBA - pull down and analysed as described above .

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A WT and R105AD106A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, at the same time cells were untreated (UT) or treated with TSQ for 6 hrs. Cells were fractionated into cytosol (C) and crude membrane (M). Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. B Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.


E HT29 cells were stimulated with TSI , ± NSA ( 500 nM ) , or left untreated ( UT ) for 16 hrs , followed by cellular fractionation . Fractions were analysed by BN - or SDS - PAGE , Western blot and probed with the indicated antibodies . Representative of three independent experiments . F Cell lysates from ( E ) were subjected to UBA - pulldown and analysed as described above .

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E HT29 cells were stimulated with TSI, ± NSA (500 nM), or left untreated (UT) for 16 hrs, followed by cellular fractionation. Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. F Cell lysates from (E) were subjected to UBA-pulldown and analysed as described above.


A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline , cells were untreated or treated with TSQ for 6 hrs ( please note that the same WT control was used in Fig . EV3A ) . Cell death was measured by PI staining based on flow cytometry . Data are plotted as mean ± SEM of three independent experiments . B Cellular fractions from ( A ) were analysed by Western blot from BN - PAGE or SDS - PAGE using antibodies as indicated . The same WT control was used in Fig . 4A . Representative of three independent experiments . C Cell lysates from ( A ) were subjected to UBA - pull down and analysed as described above .

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A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, cells were untreated or treated with TSQ for 6 hrs (please note that the same WT control was used in Fig. EV3A). Cell death was measured by PI staining based on flow cytometry. Data are plotted as mean ± SEM of three independent experiments. B Cellular fractions from (A) were analysed by Western blot from BN-PAGE or SDS-PAGE using antibodies as indicated. The same WT control was used in Fig. 4A. Representative of three independent experiments. C Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.


D N - FLAG MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline overnight , and cells were treated with TSI for indicated time , followed by UBA - pull down . Representative of three independent experiments . E N - FLAG MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline for 16 hrs . Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline . Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 ( BAF ) , PS341 ( PS ) or left untreated ( UT ) . IDN - 6556 was added to all conditions to block apoptosis . Cells were collected 0 , 2 , 4 , 6 hrs after medium replacement , followed by UBA - pull down . Representative of three independent experiments .

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D N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline overnight, and cells were treated with TSI for indicated time, followed by UBA-pull down. Representative of three independent experiments. E N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 16 hrs. Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline. Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 (BAF), PS341 (PS) or left untreated (UT). IDN-6556 was added to all conditions to block apoptosis. Cells were collected 0, 2, 4, 6 hrs after medium replacement, followed by UBA-pull down. Representative of three independent experiments.


B WT and 4KR mutant MLKL were inducibly expressed in Mlkl - / - MDFs by doxycycline for 6 hrs and cells were untreated ( UT ) or treated with TSI , followed by UBA - pull down . Representative of three independent experiments .

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B WT and 4KR mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 6 hrs and cells were untreated (UT) or treated with TSI, followed by UBA-pull down. Representative of three independent experiments.


D MLKL - / - HT29 cells stably transfected with constructs encoding human MLKL - USP21 and MLKL - USP21C221R , were treated with doxycycline , NSA ( 1 μM ) , TSI or combinations thereof ( added simultaneously ) . Sytox Green positive cells were quantified in real time by live cell imaging . Data are plotted as mean ± SEM of six independent experiments . ( A red dashed line is shown to highlight the delay in death kinetics upon treatment with NSA ) . E Human MLKL - USP21 and MLKL - USP21C221R were inducibly expressed in MLKL - / - HT29 cells by doxycycline ( 10 ng / mL ) for 16 hrs , TSI was added for the indicated times but all samples were collected 25 hrs post - induction , followed by UBA - pulldown . Representative of three independent experiments .

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D MLKL-/- HT29 cells stably transfected with constructs encoding human MLKL-USP21 and MLKL-USP21C221R, were treated with doxycycline, NSA (1 μM), TSI or combinations thereof (added simultaneously). Sytox Green positive cells were quantified in real time by live cell imaging. Data are plotted as mean ± SEM of six independent experiments. (A red dashed line is shown to highlight the delay in death kinetics upon treatment with NSA). E Human MLKL-USP21 and MLKL-USP21C221R were inducibly expressed in MLKL-/- HT29 cells by doxycycline (10 ng/mL) for 16 hrs, TSI was added for the indicated times but all samples were collected 25 hrs post-induction, followed by UBA-pulldown . Representative of three independent experiments.