An example trace showing the addition of Cy3 - labeled DNA target and the first binding event . Δτon is indicated .

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An example trace showing the addition of Cy3-labeled DNA target and the first binding event. Δτon is indicated.


Scatter plot showing the on - target binding rate ( kon - target ) for each construct . Binding rate decreases moderately with increasing number of adjacent PAMs . The values are averages of four measurements over three days . Error bars represent standard error of the mean . kon - target was calculated using the equation kon - target = 1 / ( CxΔτon ) where C is the molarity of DNA target in the channel .

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Scatter plot showing the on-target binding rate (kon-target) for each construct. Binding rate decreases moderately with increasing number of adjacent PAMs. The values are averages of four measurements over three days. Error bars represent standard error of the mean. kon-target was calculated using the equation kon-target=1/ (CxΔτon) where C is the molarity of DNA target in the channel.


E . Microscale thermophoresis binding experiment for PlexA1 - 4 - mVenus and Sema1becto ( red ) or Sema1becto - mutA ( blue ) or Sema1becto - mutB ( green , ) or Sema1becto - mutA + B ( orange ) . The Sema1becto wild - type binding results are as reported previously ( Rozbesky et al . , 2019 ) . All data were collected at the same time , error bars represent s . d . of three technical replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Microscale thermophoresis binding experiment for PlexA1-4-mVenus and Sema1becto (red) or Sema1becto-mutA (blue) or Sema1becto-mutB (green,) or Sema1becto-mutA+B (orange). The Sema1becto wild-type binding results are as reported previously (Rozbesky et al., 2019). All data were collected at the same time, error bars represent s.d. of three technical replicates.


A . FRET - FLIM in live COS - 7 cells shows cell surface PlexA - Sema1b cis interaction . Sema1b mutants reveal that both head - to - head and side - on orientations of the PlexA - Sema1b complex are involved in the cis interaction . The cis interaction between Sema1a and PlexA is not observed . The boxcharts represent the average lifetime . Box limits indicate the 25th and 75th percentiles , centred lines show the median , squares represent sample means , whiskers extend 1 . 5 - fold the interquartile range from the 25th and 75th percentiles , p - value was calculated by one - way analysis of variance ( ANOVA ) . * P < 0 . 05 , * * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. FRET-FLIM in live COS-7 cells shows cell surface PlexA-Sema1b cis interaction. Sema1b mutants reveal that both head-to-head and side-on orientations of the PlexA-Sema1b complex are involved in the cis interaction. The cis interaction between Sema1a and PlexA is not observed. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). *P<0.05, ***P<0.001


B . Representative negative stain class averages of PlexAecto correlate well with the ring - like conformation of mouse PlxnA1 . The ring - like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue ( N terminus ) to red ( C terminus ) . Scale bar , 10 nm .

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B. Representative negative stain class averages of PlexAecto correlate well with the ring-like conformation of mouse PlxnA1. The ring-like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue (N terminus) to red (C terminus). Scale bar, 10 nm.


E . In vitro FRET - FLIM measurement of the PlexA - FRET Biosensor shows that the PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation . The boxcharts represent the average lifetime . Box limits indicate the 25th and 75th percentiles , centred lines show the median , squares represent sample means , whiskers extend 1 . 5 - fold the interquartile range from the 25th and 75th percentiles , p - value was calculated by one - way analysis of variance ( ANOVA ) . * * * P < 0 . 001 .

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

E. In vitro FRET-FLIM measurement of the PlexA-FRET Biosensor shows that the PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). ***P<0.001.


F . Relative change of COS - 7 cells surface area upon treatment with Sema1a - Fc . Cells expressing PlexAFL - mClover or PlexAFL - mCover and Sema1b - mRuby2 wild - type or mutants were treated with purified Sema1a - Fc at a final concentration of 5 . 8 µM . Images were acquired every minute for 30 minutes . Cell surface area was calculated using ImageJ before and after stimulation . Data are presented as means ± sem . * P < 0 . 05 , * * * P < 0 . 001 , p - value calculated by one - way analysis of variance ( ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Relative change of COS-7 cells surface area upon treatment with Sema1a-Fc. Cells expressing PlexAFL-mClover or PlexAFL-mCover and Sema1b-mRuby2 wild-type or mutants were treated with purified Sema1a-Fc at a final concentration of 5.8 µM. Images were acquired every minute for 30 minutes. Cell surface area was calculated using ImageJ before and after stimulation. Data are presented as means ± sem. *P<0.05, ***P<0.001, p-value calculated by one-way analysis of variance (ANOVA).


A . Cultured dorsal root ganglion ( DRG ) neurons from E12 . 5 Sema6A knockout ( KO ) embryos were transfected with EGFP , EGFP - tagged wild - type ( WT ) Sema6A ( Sema6A - WT ) or different EGFP - tagged Sema6A mutants ( Sema6A - mutA , Sema6A - mutB , and Sema6A - mutA + B ) at 1 day in vitro ( DIV1 ) and treated with purified Sema6A - Fc at a concentration of 75 nM for 1 hour at 37 ° C at DIV4 . Transfection of EGFP was used as a control ( full collapse ) . DRG neurons from Sema6A KO embryos acquired sensitivity to Sema6A and growth cone collapse was observed in three independent experiments . Transfection of Sema6A - WT , Sema6A - mutA and Sema6A - mutB but not of EGFP or Sema6A - mutantA + B prevented Sema6A - mediated growth cone collapse ( Fisher ' s exact test p < 0 . 0001 ) . Quantification of growth cone collapse was performed using a growth cone morphology matrix ( lower part panel A ) . Data is presented as percentage of morphologically distinct growth cones , divided ( represented by the dotted line ) in two categories , from uncollapsed ( 1 - 4 ) to fully collapsed ( 5 - 8 ) . Totally , 20 - 40 growth cones were analyzed for each condition per experiment ( n = 3 experiments ) . Scale bar 20 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cultured dorsal root ganglion (DRG) neurons from E12.5 Sema6A knockout (KO) embryos were transfected with EGFP, EGFP-tagged wild-type (WT) Sema6A (Sema6A-WT) or different EGFP-tagged Sema6A mutants (Sema6A-mutA, Sema6A-mutB, and Sema6A-mutA+B) at 1 day in vitro (DIV1) and treated with purified Sema6A-Fc at a concentration of 75 nM for 1 hour at 37°C at DIV4. Transfection of EGFP was used as a control (full collapse). DRG neurons from Sema6A KO embryos acquired sensitivity to Sema6A and growth cone collapse was observed in three independent experiments. Transfection of Sema6A-WT, Sema6A-mutA and Sema6A-mutB but not of EGFP or Sema6A-mutantA+B prevented Sema6A-mediated growth cone collapse (Fisher's exact test p<0.0001). Quantification of growth cone collapse was performed using a growth cone morphology matrix (lower part panel A). Data is presented as percentage of morphologically distinct growth cones, divided (represented by the dotted line) in two categories, from uncollapsed (1-4) to fully collapsed (5-8). Totally, 20-40 growth cones were analyzed for each condition per experiment (n=3 experiments). Scale bar 20 µm.


B . Representative images of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A - Fc and visualized with immunofluorescent staining to detect GFP ( green ) and phalloidin ( red ) . Average intensity images for all different constructs are shown in grey color . Scale bar 20 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

B. Representative images of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A-Fc and visualized with immunofluorescent staining to detect GFP (green) and phalloidin (red). Average intensity images for all different constructs are shown in grey color. Scale bar 20 µm.


A . Representative H & E staining and immunohistochemical analysis of the androgen receptor ( AR ) in the dorsolateral prostate ( DLP ) of PtenL2 / L2 and Pten ( i ) pe - / - mice , sham - operated ( sham ) or castrated ( CTX ) at 3 months after gene inactivation ( AGI ) , and analyzed at 1 month after surgery . Scale bars : 100 µm . N = 3 mice per condition .

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A. Representative H&E staining and immunohistochemical analysis of the androgen receptor (AR) in the dorsolateral prostate (DLP) of PtenL2/L2 and Pten(i)pe-/- mice, sham-operated (sham) or castrated (CTX) at 3 months after gene inactivation (AGI), and analyzed at 1 month after surgery. Scale bars: 100 µm. N=3 mice per condition.


F . Violin plots depicting transcript levels of androgen - responsive genes in luminal - C cells of sham and CTX Pten ( i ) pe - / - mice . P - values were determined by Wilcoxon rank sum test .

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F. Violin plots depicting transcript levels of androgen-responsive genes in luminal-C cells of sham and CTX Pten(i)pe-/- mice. P-values were determined by Wilcoxon rank sum test.


H . Violin plots depicting the transcript levels of inflammation - related genes in luminal - C cells of sham and CTX Pten ( i ) pe - / - mice . P - values were determined by Wilcoxon rank sum test .

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H. Violin plots depicting the transcript levels of inflammation-related genes in luminal-C cells of sham and CTX Pten(i)pe-/- mice. P-values were determined by Wilcoxon rank sum test.


J , K . Violin plots depicting the transcript levels of hypoxia - related genes ( J ) and plasticity - related genes ( K ) in luminal - C cells of sham and CTX Pten ( i ) pe - / - mice . P - values were determined by Wilcoxon rank sum test .

J, K. Violin plots depicting the transcript levels of hypoxia-related genes (J) and plasticity-related genes (K) in luminal-C cells of sham and CTX Pten(i)pe-/- mice. P-values were determined by Wilcoxon rank sum test.


C . Gland areas in DLP of sham and CTX control and Pten / Hif1a ( i ) pe - / - mice . Five glands per mouse were quantified . N = 3 mice / condition .

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C. Gland areas in DLP of sham and CTX control and Pten/Hif1a(i)pe-/- mice. Five glands per mouse were quantified. N=3 mice/condition.


G . Representative immunohistochemical detection of cleaved caspase 3 ( CC3 ) in DLP of Pten ( i ) pe - / - and Pten / Hif1a ( i ) pe - / - mice , sham or CTX at 3 months AGI , and analyzed 1 or 1 , 2 , 3 and 30 days after surgery , respectively . N = 3 mice / condition .

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G. Representative immunohistochemical detection of cleaved caspase 3 (CC3) in DLP of Pten(i)pe-/- and Pten/Hif1a(i)pe-/- mice, sham or CTX at 3 months AGI, and analyzed 1 or 1, 2, 3 and 30 days after surgery, respectively. N=3 mice/condition.


I . Western blot analysis of CC3 in protein lysates of organoids generated from prostates of Pten ( i ) pe - / - and Pten / Hif1a ( i ) pe - / - mice at 3 months AGI , and treated with enzalutamide ( Enz ) for 24 hours at the indicated concentrations . β - actin was used as a loading control .

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I. Western blot analysis of CC3 in protein lysates of organoids generated from prostates of Pten(i)pe-/- and Pten/Hif1a(i)pe-/- mice at 3 months AGI, and treated with enzalutamide (Enz) for 24 hours at the indicated concentrations. β-actin was used as a loading control.


B . Representative H & E staining of DLP of Pten / Hif1a ( i ) pe - / - mice sham and CTX at 3 months AGI and analyzed at 5 months after surgery . N = 4 - 5 mice / condition . C . Gland areas in DLP of control and Pten / Hif1a ( i ) pe - / - mice , sham and CTX at 3 months AGI and analyzed at 5 months after surgery . N = 3 - 4 mice / condition . Five glands per mouse were quantified . Data presented are mean , and error bars correspond to SEM .

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B. Representative H&E staining of DLP of Pten/Hif1a(i)pe-/- mice sham and CTX at 3 months AGI and analyzed at 5 months after surgery. N=4-5 mice/condition. C. Gland areas in DLP of control and Pten/Hif1a(i)pe-/- mice, sham and CTX at 3 months AGI and analyzed at 5 months after surgery. N=3-4 mice/condition. Five glands per mouse were quantified. Data presented are mean, and error bars correspond to SEM.


E . Representative immunohistochemical detection of AR and TROP2 in DLP of Pten / Hif1a ( i ) pe - / - mice , sham and CTX at 3 months AGI and analyzed at 5 months after surgery . N = 3 - 4 mice / condition .

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E. Representative immunohistochemical detection of AR and TROP2 in DLP of Pten/Hif1a(i)pe-/- mice, sham and CTX at 3 months AGI and analyzed at 5 months after surgery. N = 3-4 mice/condition.


D . Representative western blot analysis of CPARP and CC3 in human C4 - 2B PCa cells treated for 24 h with PX - 478 , increasing concentrations of enzalutamide ( Enz ) , or a combination of both . Beta - actin was used as loading control .

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D. Representative western blot analysis of CPARP and CC3 in human C4-2B PCa cells treated for 24 h with PX-478, increasing concentrations of enzalutamide (Enz), or a combination of both. Beta-actin was used as loading control.


F . Representative H & E staining of DLP of sham and CTX Pten ( i ) pe - / - mice , treated with vehicle or PX - 478 , as depicted in ( E ) . Scale bar : 100 µm . N = 3 - 4 mice / condition .

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F. Representative H&E staining of DLP of sham and CTX Pten(i)pe-/- mice, treated with vehicle or PX-478, as depicted in (E). Scale bar: 100 µm. N=3-4 mice/condition.


H . Gland areas of DLP of sham and CTX Pten ( i ) pe - / - mice , treated with vehicle or PX - 478 , as depicted in ( E ) . N = 3 - 4 mice / condition . Data presented are mean ± SEM . Five glands per mouse were quantified ,

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H. Gland areas of DLP of sham and CTX Pten(i)pe-/- mice, treated with vehicle or PX-478, as depicted in (E). N=3-4 mice/condition. Data presented are mean ± SEM. Five glands per mouse were quantified,


B . DIC microscopic image of WTSOD12SH diluted to 100 µM in HEPES buffer , pH 7 . 4 and 100 mM NaCl and incubated 37 ° C , 180 RPM for 30 min shows no droplet formation ; scale bar : 100 µm ; inset shows Alexa Fluor 488 maleimide labelled WTSOD12SH ;

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B. DIC microscopic image of WTSOD12SH diluted to 100 µM in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated 37 °C, 180 RPM for 30 min shows no droplet formation; scale bar: 100 µm; inset shows Alexa Fluor 488 maleimide labelled WTSOD12SH;


C . Solution turbidity plot ( absorbance at 600 nm ) shows that WTSOD12SH ( at concentrations ranging from 25 to 200 µM does not undergo LLPS in presence or absence of heparin , LLPS inducer ; data is represented as mean ± SD ( from 3 biological replicates ) .

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C. Solution turbidity plot (absorbance at 600 nm) shows that WTSOD12SH (at concentrations ranging from 25 to 200 µM does not undergo LLPS in presence or absence of heparin, LLPS inducer; data is represented as mean ± SD (from 3 biological replicates).


D . DIC microscopic image of ApoSOD12SH under condensate inducing conditions ( 37  C , 180 RPM ) at a concentration of 100 µM incubated 100 mM NaCl ; scale bar : 100 µm ( inset on bottom left shows Alexa Fluor 488 maleimide labeled protein condensates ;

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D. DIC microscopic image of ApoSOD12SH under condensate inducing conditions (37 C, 180 RPM) at a concentration of 100 µM incubated 100 mM NaCl; scale bar:100 µm (inset on bottom left shows Alexa Fluor 488 maleimide labeled protein condensates;


E . Fluorescent microscopic images of Alexa Fluor 488 maleimide labeled ApoSOD12SH condensates diluted in HEPES buffer , pH 7 . 4 and 100 mM NaCl and incubated ( 180 RPM , 37  C ) in absence ( left ) and presence ( right ) of heparin at 30 min time -

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E. Fluorescent microscopic images of Alexa Fluor 488 maleimide labeled ApoSOD12SH condensates diluted in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated (180 RPM, 37 C) in absence (left) and presence (right) of heparin at 30 min time-point


F . Solution turbidity measurements with ApoSOD12SH ( absorbance at 600 nm ) shows that while in the absence of heparin the turbidity increases with time , the presence of heparin results in faster LLPS ;

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F. Solution turbidity measurements with ApoSOD12SH (absorbance at 600 nm) shows that while in the absence of heparin the turbidity increases with time, the presence of heparin results in faster LLPS;


G . Comparison of ApoSOD12SH droplet numbers per microscopic field of view ( view area 0 . 001963 mm2 ) calculated from 5 different images of droplets incubated with and without heparin at 2 h timepoint .

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G. Comparison of ApoSOD12SH droplet numbers per microscopic field of view (view area 0.001963 mm2) calculated from 5 different images of droplets incubated with and without heparin at 2 h timepoint.


H . Liquid nature of ApoSOD12SH showing droplet fusion and surface

H. Liquid nature of ApoSOD12SH showing droplet fusion and surface wetting


I . Amplitude normalized FCS curves of ApoSOD12SH ( in blue ) of dilute phase ( DP ) and condensed phase ( CP ) . Intense color indicates CP and light variant indicates DP . The increase in τD ( as shown by the arrow ) translates to a decrease in

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

I. Amplitude normalized FCS curves of ApoSOD12SH (in blue) of dilute phase (DP) and condensed phase (CP). Intense color indicates CP and light variant indicates DP. The increase in τD (as shown by the arrow) translates to a decrease in D


J . Scheme depicting that presence of Zn and not Cu in pre - incubation mixture inhibits LLPS . K . DIC microscopic images of ApoSOD12SH in presence of Zn ( top ) showing no droplet formation and Cu ( below ) showing the presence of

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J. Scheme depicting that presence of Zn and not Cu in pre-incubation mixture inhibits LLPS. K. DIC microscopic images of ApoSOD12SH in presence of Zn (top) showing no droplet formation and Cu (below) showing the presence of droplets


L . Solution turbidity plot ( absorbance at 600 nm ) of ApoSOD12SH subjected to increasing concentrations of Cu and Zn ; turbidity decreases in a dose dependent manner for Zn while no significant change is observed in presence of Cu ;

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L. Solution turbidity plot (absorbance at 600 nm) of ApoSOD12SH subjected to increasing concentrations of Cu and Zn; turbidity decreases in a dose dependent manner for Zn while no significant change is observed in presence of Cu;


A . DIC microscopic images of I113T SOD12SH ( top ) and G85R SOD12SH ( bottom ) droplets incubated in the absence and presence of Zn and Cu respectively ( scale bar : 100 µm ) ; insets on bottom left show Alexa Fluor 488 maleimide labeled protein condensates for I113T SOD12SH and G85R SOD12SH respectively ; scale bar : 5 µm . Droplet dissolution was observed for I113T SOD12SH on addition of Zn while droplets persisted on addition of Cu . Red arrowhead indicates fusion of liquid droplets .

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A. DIC microscopic images of I113T SOD12SH (top) and G85R SOD12SH (bottom) droplets incubated in the absence and presence of Zn and Cu respectively (scale bar: 100 µm); insets on bottom left show Alexa Fluor 488 maleimide labeled protein condensates for I113T SOD12SH and G85R SOD12SH respectively; scale bar: 5 µm. Droplet dissolution was observed for I113T SOD12SH on addition of Zn while droplets persisted on addition of Cu. Red arrowhead indicates fusion of liquid droplets.


B . Amplitude normalized FCS curves of I113T SOD12SH ( in green ) and G85R SOD12SH ( in grey ) of dilute phase ( DP ) and condensed phase ( CP ) showing an increase in diffusion time from DP to CP . The intense color indicates CP and light variant indicates DP .

B. Amplitude normalized FCS curves of I113T SOD12SH (in green) and G85R SOD12SH (in grey) of dilute phase (DP) and condensed phase (CP) showing an increase in diffusion time from DP to CP. The intense color indicates CP and light variant indicates DP.


C . On metal cofactor removal , ApoG37R SOD12SH formed condensates which ceased to exist when protein was incubated with Zn , while condensates persisted on Cu addition ( left to right ) ;

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C. On metal cofactor removal, ApoG37R SOD12SH formed condensates which ceased to exist when protein was incubated with Zn, while condensates persisted on Cu addition (left to right);


B . Plot of the loge of the hydrodynamic radius ( rH ) versus the log e of the number of residues in the polypeptide chain . The line fitted to these data for the native folded proteins ( yellow dashed ) has a slope of 0 . 29 ± 0 . 02 and a y - axis intercept of 1 . 56 ± 0 . 1 , while the other fitted to the chemically denatured protein ( grey dashed ) data has a slope of 0 . 57 ± 0 . 02 and a y - axis intercept of 0 . 79 ± 0 . 07 . Literature data have been used for folded and chemically denatured proteins while we employed FCS to calculate the rH of all SOD12SH variants with ( solid shapes ) and without Zn ( hollow shapes ) . The hollow grey circle of G85R SOD12SH is directly under hollow green I113T SOD12SH in the log - log 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

B. Plot of the loge of the hydrodynamic radius (rH) versus the log e of the number of residues in the polypeptide chain. The line fitted to these data for the native folded proteins (yellow dashed) has a slope of 0.29 ± 0.02 and a y-axis intercept of 1.56 ± 0.1, while the other fitted to the chemically denatured protein (grey dashed) data has a slope of 0.57 ± 0.02 and a y-axis intercept of 0.79 ± 0.07. Literature data have been used for folded and chemically denatured proteins while we employed FCS to calculate the rH of all SOD12SH variants with (solid shapes) and without Zn (hollow shapes). The hollow grey circle of G85R SOD12SH is directly under hollow green I113T SOD12SH in the log-log plot.


C . Variation of diffusion coefficients of de - metalated protein variants determined from FCS measurement with increasing Zn concentration ( inset shows a comparison of binding constants between mutants and Zn ; ApoSOD12SH and ApoG37R SOD12SH has a higher binding affinity to Zn than ApoI113T SOD12SH and ApoG85R SOD12SH ) .

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 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

C. Variation of diffusion coefficients of de-metalated protein variants determined from FCS measurement with increasing Zn concentration (inset shows a comparison of binding constants between mutants and Zn; ApoSOD12SH and ApoG37R SOD12SH has a higher binding affinity to Zn than ApoI113T SOD12SH and ApoG85R SOD12SH).


D . Disordered / extended conformation content decreases in ApoSOD12SH and ApoI113T SOD12SH with the addition of Zn calculated from FTIR spectra ;

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

D. Disordered/ extended conformation content decreases in ApoSOD12SH and ApoI113T SOD12SH with the addition of Zn calculated from FTIR spectra;


C . Salt - bridge analysis of ApoG85R SOD12SH ( dist . cutoff < 0 . 5 nm = salt - bridge ) .

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C. Salt-bridge analysis of ApoG85R SOD12SH (dist. cutoff <0.5 nm = salt-bridge).


C . Upper panel shows fluorescence images of the maturation of Alexa Fluor 488 labeled ApoSOD12SH droplets with time , samples were incubated at 37  C up to ~ 25 hours . Images are taken at indicated time point : 0 * hour ( the time after incubation required for droplet formation ) , 12 hours and > 24 hours ; images below show magnified scale bar : 5 µm . The middle panel shows magnified view of the marked region from corresponding images on the upper panel . Red arrowhead indicates matured ( solid ) portion inside the droplet . Lower panel shows coexistence of liquid and solid phase inside droplet ; scale bar : 10 µm . Intensity plot ( bottom right ) shows the distribution of high intensity region within matured droplet .

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C. Upper panel shows fluorescence images of the maturation of Alexa Fluor 488 labeled ApoSOD12SH droplets with time, samples were incubated at 37 C up to ~25 hours. Images are taken at indicated time point: 0* hour (the time after incubation required for droplet formation), 12 hours and >24 hours; images below show magnified scale bar: 5 µm. The middle panel shows magnified view of the marked region from corresponding images on the upper panel. Red arrowhead indicates matured (solid) portion inside the droplet. Lower panel shows coexistence of liquid and solid phase inside droplet; scale bar: 10 µm. Intensity plot (bottom right) shows the distribution of high intensity region within matured droplet.


D . Diffusion coefficient obtained from point FCS measurement at different regions of the droplet during maturation course ; 0 * ( x - axis label ) indicates the time after incubation required for droplet formation .

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D. Diffusion coefficient obtained from point FCS measurement at different regions of the droplet during maturation course; 0* (x-axis label) indicates the time after incubation required for droplet formation.


E . ThT fluorescence assay plot showing aggregation kinetics of all SOD12SH variants . ApoSOD12SH , I113T SOD12SH and G85R SOD12SH show high ThT fluorescence .

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E. ThT fluorescence assay plot showing aggregation kinetics of all SOD12SH variants. ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show high ThT fluorescence.


F . AFM micrographs of ApoSOD12SH , I113T SOD12SH and G85R SOD12SH show fibrillar aggregates after prolonged incubation at 37 ℃ , 180 RPM in absence of Zn ( left panel ) ; AFM micrographs of ApoSOD12SH , I113T SOD12SH and G85R SOD12SH incubated with Zn ( at 1 : 5 protein : Zn molar ratio ) show presence of oligomers for ApoSOD12SH , I113T SOD12SH and short fibrils for G85R SOD12SH ( right panel ) ;

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F. AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show fibrillar aggregates after prolonged incubation at 37 ℃, 180 RPM in absence of Zn (left panel); AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH incubated with Zn (at 1:5 protein: Zn molar ratio) show presence of oligomers for ApoSOD12SH, I113T SOD12SH and short fibrils for G85R SOD12SH (right panel);


G . Cell proliferation of SHSY5Y human neuroblastoma cells assessed by MTT assay post 12 h treatment with 5 µM ApoSOD12SH , I113T SOD12SH and G85R SOD12SH condensates and aggregates ( fibrils ) . Cell culture medium ( DMEM ) was used as negative control and untreated SHSY5Y cells were used as positive control .

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G. Cell proliferation of SHSY5Y human neuroblastoma cells assessed by MTT assay post 12 h treatment with 5 µM ApoSOD12SH, I113T SOD12SH and G85R SOD12SH condensates and aggregates (fibrils). Cell culture medium (DMEM) was used as negative control and untreated SHSY5Y cells were used as positive control.


H . Dot plots showing flow cytometric analysis of annexin V and propidium iodide ( PI ) staining of apoptotic SHSY5Y neuroblastoma cells following a 12 h treatment with 5 µM ApoSOD12SH condensates , I113T SOD12SH condensates , G85R SOD12SH condensates , ApoSOD12SH fibrils , I113T SOD12SH fibrils and G85R SOD12SH fibrils respectively ( see Methods for details ) . High cytotoxicity was observed for SOD12SH condensates .

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

H. Dot plots showing flow cytometric analysis of annexin V and propidium iodide (PI) staining of apoptotic SHSY5Y neuroblastoma cells following a 12 h treatment with 5 µM ApoSOD12SH condensates, I113T SOD12SH condensates, G85R SOD12SH condensates, ApoSOD12SH fibrils, I113T SOD12SH fibrils and G85R SOD12SH fibrils respectively (see Methods for details). High cytotoxicity was observed for SOD12SH condensates.


( b ) THPN - induced cell death is prevented by the autophagic inhibitors wortmannin ( 100 nM ) and chloroquine ( 20 μM ) but not by the apoptotic inhibitor zVAD ( 20 μM ) or the necrotic inhibitors butylated hydroxyanisole ( 100 μM ) and necrostatin - 1 ( 30 μM ) . The cells were pretreated with each inhibitor for 1 h separately before the THPN treatment ( 20 μM , 48 h ) . Ctrl , control .

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

(b) THPN-induced cell death is prevented by the autophagic inhibitors wortmannin (100 nM) and chloroquine (20 μM) but not by the apoptotic inhibitor zVAD (20 μM) or the necrotic inhibitors butylated hydroxyanisole (100 μM) and necrostatin-1 (30 μM). The cells were pretreated with each inhibitor for 1 h separately before the THPN treatment (20 μM, 48 h). Ctrl, control.


( c ) THPN induces autophagy in melanoma cells . Top , THPN ( 20 μM , 24 h ) induces a change in the localization of GFP - LC3 fusion proteins from a ubiquitous , diffuse pattern to cytoplasmic puncta . Serum starvation ( SS , 24 h ) was used as a positive control ( scale bars , 10 μm ) . Middle , the GFP - LC3 - positive cells with puncta were calculated from five randomly picked regions . Bottom , the conversion of LC3 - I to LC3 - II induced by THPN was determined by western blotting . Images of full blots are shown in Supplementary Figure 7 . All of the data are presented as the mean ± s . e . m . of three independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; NS , not significant .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O 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) THPN induces autophagy in melanoma cells. Top, THPN (20 μM, 24 h) induces a change in the localization of GFP-LC3 fusion proteins from a ubiquitous, diffuse pattern to cytoplasmic puncta. Serum starvation (SS, 24 h) was used as a positive control (scale bars, 10 μm). Middle, the GFP-LC3-positive cells with puncta were calculated from five randomly picked regions. Bottom, the conversion of LC3-I to LC3-II induced by THPN was determined by western blotting. Images of full blots are shown in Supplementary Figure 7. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01, ***P 0.001; NS, not significant.


( a ) THPN induces TR3 translocation from the cytoplasm to the mitochondria , detected by western blotting for the various cell fractionations ( top ) and by confocal microscopy ( bottom ) . The cells were treated with THPN at the times indicated and detected by TR3 ( red ) and Hsp60 ( green ) . The nuclei were stained with DAPI ( blue ) ( scale bars , 10 μm ) . PARP , Tubulin and Hsp60 were used as loading controls for nuclear extracts ( NE ) , cytoplasmic extracts ( CE ) and mitochondrial fractions ( Mito ) , respectively . IB , immunoblotting .

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 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 O O O O O O O O

(a) THPN induces TR3 translocation from the cytoplasm to the mitochondria, detected by western blotting for the various cell fractionations (top) and by confocal microscopy (bottom). The cells were treated with THPN at the times indicated and detected by TR3 (red) and Hsp60 (green). The nuclei were stained with DAPI (blue) (scale bars, 10 μm). PARP, Tubulin and Hsp60 were used as loading controls for nuclear extracts (NE), cytoplasmic extracts (CE) and mitochondrial fractions (Mito), respectively. IB, immunoblotting.


( b ) TR3 mutant Flag - ΔNLS but not Flag - NLS promotes the induction of autophagy in response to THPN treatment . The siRNA - resistant versions of Flag - ΔNLS and Flag - NLS were transfected into TR3 - knockdown A375 cells . Top , the cells cotransfected with GFP - LC3 were further treated with THPN for 24 h before microscopic analysis ( scale bars , 10 μm ) . Middle , the conversion of LC3 - I to LC3 - II was analyzed by western blotting . The rate of cell survival was determined after THPN treatment for 48 h ( bottom ) . ' - ' represents vehicle treatment only .

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

(b) TR3 mutant Flag-ΔNLS but not Flag-NLS promotes the induction of autophagy in response to THPN treatment. The siRNA-resistant versions of Flag-ΔNLS and Flag-NLS were transfected into TR3-knockdown A375 cells. Top, the cells cotransfected with GFP-LC3 were further treated with THPN for 24 h before microscopic analysis (scale bars, 10 μm). Middle, the conversion of LC3-I to LC3-II was analyzed by western blotting. The rate of cell survival was determined after THPN treatment for 48 h (bottom). '-' represents vehicle treatment only.


( c ) Nix assists the TR3 mitochondrial targeting . Cells with Nix , Pink1 and Bnip3 knockdown were treated with THPN ( 20 μM , 6 h ) . Top , mitochondrial TR3 was assayed by western blotting . Nix knockdown abolishes the THPN - induced ( 20 μM , 24 h ) formation of LC3 foci ( middle and bottom ; scale bars , 10 μm ) . Images of full blots are shown in Supplementary Figure 7 . All of the data are presented as the mean ± s . e . m . of three independent experiments . * * P 0 . 01 ; NS , not significant ; ctrl , control .

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-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 O O O O O O O O O O O O

(c) Nix assists the TR3 mitochondrial targeting. Cells with Nix, Pink1 and Bnip3 knockdown were treated with THPN (20 μM, 6 h). Top, mitochondrial TR3 was assayed by western blotting. Nix knockdown abolishes the THPN-induced (20 μM, 24 h) formation of LC3 foci (middle and bottom; scale bars, 10 μm). Images of full blots are shown in Supplementary Figure 7. All of the data are presented as the mean ± s.e.m. of three independent experiments. **P 0.01; NS, not significant; ctrl, control.


( b ) Site C of LBD is critical for Nix binding , as determined by the in vitro pull - down assay . The purified bead - bound GST or GST - Nix proteins were incubated with different mutants as indicated and THPN ( 20 μM ) for 3 h ; the proteins were then analyzed by western blotting . IB , immunoblotting .

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(b) Site C of LBD is critical for Nix binding, as determined by the in vitro pull-down assay. The purified bead-bound GST or GST-Nix proteins were incubated with different mutants as indicated and THPN (20 μM) for 3 h; the proteins were then analyzed by western blotting. IB, immunoblotting.


( e ) DPDO cannot facilitate Nix binding to TR3 . Top , the structure of DPDO . Middle , DPDO binding could not form the interacting surface for Nix , unlike THPN . The structures of Arg563 and Ser553 are the same as those in the native structure . Bottom , DPDO does not enhance the interaction between TR3 and Nix . Different plasmids were transfected into A375 cells as indicated , and cells were treated with DPDO ( 20 μM , 3 h ) . The TR3 interaction with Nix was determined by co - immunoprecipitation . Images of full blots are shown in Supplementary Figure 7 . IP , immunoprecipitation ; HA , hemagglutinin .

O O O B-CONTROLLED_VAR 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 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 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

(e) DPDO cannot facilitate Nix binding to TR3. Top, the structure of DPDO. Middle, DPDO binding could not form the interacting surface for Nix, unlike THPN. The structures of Arg563 and Ser553 are the same as those in the native structure. Bottom, DPDO does not enhance the interaction between TR3 and Nix. Different plasmids were transfected into A375 cells as indicated, and cells were treated with DPDO (20 μM, 3 h). The TR3 interaction with Nix was determined by co-immunoprecipitation. Images of full blots are shown in Supplementary Figure 7. IP, immunoprecipitation; HA, hemagglutinin.


( a ) THPN induces the dissipation of ΔΨm in both time - and dose - dependent manners . The compromised ΔΨm is shifted to the right upon THPN treatment in flow cytometry . ' - ' represents vehicle treatment only .

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(a) THPN induces the dissipation of ΔΨm in both time- and dose-dependent manners. The compromised ΔΨm is shifted to the right upon THPN treatment in flow cytometry. '-' represents vehicle treatment only.


( b ) Effects of different plasmids on the THPN - induced dissipation of ΔΨm . Top , endogenous TR3 was knocked down by shRNA , and the siRNA - resistant version of TR3 , TR3 - NLS , TR3 - ΔNLS or TAD ( Supplementary Fig . 2c ) were separately transfected into the cells as indicated . Bottom , similarly , the siRNA - resistant version of Nix or ΔTM were separately transfected into Nix knockdown cells . Transfected cells were treated with THPN ( 20 μM , 12 h ) , and their ΔΨm was assessed by flow cytometry . Ctrl , control .

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 O O O O O O O O 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

(b) Effects of different plasmids on the THPN-induced dissipation of ΔΨm. Top, endogenous TR3 was knocked down by shRNA, and the siRNA-resistant version of TR3, TR3-NLS, TR3-ΔNLS or TAD (Supplementary Fig. 2c) were separately transfected into the cells as indicated. Bottom, similarly, the siRNA-resistant version of Nix or ΔTM were separately transfected into Nix knockdown cells. Transfected cells were treated with THPN (20 μM, 12 h), and their ΔΨm was assessed by flow cytometry. Ctrl, control.


( c ) ANT1 and VDAC1 are required for the THPN - induced autophagy . The ANT1 and VDAC1 shRNA - expressing plasmids , which also encoded EGFP for evaluating the transfection efficiency , were separately transfected into cells ; mCherry - LC3 was cotransfected to display autophagy . Green cells are those transfected with ANT1 or VDAC1 shRNA , and the cells with red cytoplasmic puncta are autophagic cells ( top ; scale bars , 10 μm ) .

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(c) ANT1 and VDAC1 are required for the THPN-induced autophagy. The ANT1 and VDAC1 shRNA-expressing plasmids, which also encoded EGFP for evaluating the transfection efficiency, were separately transfected into cells; mCherry-LC3 was cotransfected to display autophagy. Green cells are those transfected with ANT1 or VDAC1 shRNA, and the cells with red cytoplasmic puncta are autophagic cells (top; scale bars, 10 μm).


( d ) ANT1 and VDAC1 are required for the THPH - induced dissipation of ΔΨm and cell death . Endogenous ANT1 or VDAC1 was separately knocked down by shRNA , and the dissipation of ΔΨm was determined after THPN ( 20 μM , 12 h ) treatment ( left ) . ANT1 and VDAC1 knockdown abolishes the THPN - induced ( 20 μM , 48 h ) cell death ( right ) . All of the data are presented as the mean ± s . e . m . of three independent experiments . * * 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 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 O O O O O O O O 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) ANT1 and VDAC1 are required for the THPH-induced dissipation of ΔΨm and cell death. Endogenous ANT1 or VDAC1 was separately knocked down by shRNA, and the dissipation of ΔΨm was determined after THPN (20 μM, 12 h) treatment (left). ANT1 and VDAC1 knockdown abolishes the THPN-induced (20 μM, 48 h) cell death (right). All of the data are presented as the mean ± s.e.m. of three independent experiments. **P 0.01.


( a ) Endogenous TR3 is localized in the MIM in response to THPN stimulation . Left , isolated mitochondria from cells treated with THPN ( 20 μM , 6 h ) were incubated with proteinase K ( 20 μM and 40 μM ) for 30 min . TR3 expression was determined . Tom20 in MOM and Tim23 in MIM were used as controls . Right , the mitochondria were further subjected to submitochondrial fractionation . VDAC1 , Tim23 and Hsp60 were used to represent MOM , MIM and mitochondrial matrix protein , respectively . ' - ' represents vehicle treatment only .

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(a) Endogenous TR3 is localized in the MIM in response to THPN stimulation. Left, isolated mitochondria from cells treated with THPN (20 μM, 6 h) were incubated with proteinase K (20 μM and 40 μM) for 30 min. TR3 expression was determined. Tom20 in MOM and Tim23 in MIM were used as controls. Right, the mitochondria were further subjected to submitochondrial fractionation. VDAC1, Tim23 and Hsp60 were used to represent MOM, MIM and mitochondrial matrix protein, respectively. '-' represents vehicle treatment only.


( b ) Knockdown of Tom40 and Tom70 blocks TR3 transit to MIM . Endogenous Toms were separately knocked down by their corresponding shRNAs , and the TR3 expression of mitochondrial fractions was detected as described above . Ctrl , control .

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(b) Knockdown of Tom40 and Tom70 blocks TR3 transit to MIM. Endogenous Toms were separately knocked down by their corresponding shRNAs, and the TR3 expression of mitochondrial fractions was detected as described above. Ctrl, control.


( c ) Tom40 and Tom70 are required for the THPN - associated dissipation of ΔΨm ( left ) , conversion of LC3 - I to LC3 - II ( right and top ) and cell death ( right and bottom ) . Endogenous Toms were separately knocked down by the corresponding shRNAs . ΔΨm , LC3 - II and cell death were detected as described above . Images of full blots are shown in Supplementary Figure 7 .

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(c) Tom40 and Tom70 are required for the THPN-associated dissipation of ΔΨm (left), conversion of LC3-I to LC3-II (right and top) and cell death (right and bottom). Endogenous Toms were separately knocked down by the corresponding shRNAs. ΔΨm, LC3-II and cell death were detected as described above. Images of full blots are shown in Supplementary Figure 7.


( d ) The mitochondria were markedly eliminated after treatment with THPN for 24 h . Right , endogenous Tom40 and Tom70 were knocked down , and the amount of mitochondria was measured by the fluorescence intensity of Tom20 ( green ) . Left , the volume of mitochondria from five randomly picked regions was quantified by ImageJ software . All of the data are presented as the mean ± s . e . m . of three independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; NS , not significant .

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(d) The mitochondria were markedly eliminated after treatment with THPN for 24 h. Right, endogenous Tom40 and Tom70 were knocked down, and the amount of mitochondria was measured by the fluorescence intensity of Tom20 (green). Left, the volume of mitochondria from five randomly picked regions was quantified by ImageJ software. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01, ***P 0.001; NS, not significant.


( a ) THPN inhibits the formation of tumors in the liver and lung in a TR3 - dependent manner . B16 cells transfected with either control shRNA , TR3 shRNA or ATG7 shRNA ( 5 × 105 ) were separately injected into the lateral tail vein of 6 - week - old C57BL / 6J mice ( n = 7 ) . THPN were administered 3 d after inoculation . The tumors formed in the lung and liver are indicated ( top ; scale bars , 5 mm ) and were counted ( bottom ) . Ctrl , control .

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 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

(a) THPN inhibits the formation of tumors in the liver and lung in a TR3-dependent manner. B16 cells transfected with either control shRNA, TR3 shRNA or ATG7 shRNA (5 × 105) were separately injected into the lateral tail vein of 6-week-old C57BL/6J mice (n = 7). THPN were administered 3 d after inoculation. The tumors formed in the lung and liver are indicated (top; scale bars, 5 mm) and were counted (bottom). Ctrl, control.


( b ) Crossed Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 + / + and Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 − / − mice ( n = 12 ) were treated topically with DMBA to initiate cutaneous melanocytic neoplasms , and mice were treated with THPN as described in Online Methods . The representative images of dorsal melanomas ( top ) and the tumor numbers ( bottom ) are presented .

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

(b) Crossed Tyr:NRasQ61K;Ink4a/Arf−/−;TR3+/+ and Tyr:NRasQ61K;Ink4a/Arf−/−;TR3−/− mice (n = 12) were treated topically with DMBA to initiate cutaneous melanocytic neoplasms, and mice were treated with THPN as described in Online Methods. The representative images of dorsal melanomas (top) and the tumor numbers (bottom) are presented.


( c ) The survival curves of crossed Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 + / + and Tyr : NRasQ61K ; Ink4a / Arf − / − ; TR3 − / − mice are shown ( n > 16 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) The survival curves of crossed Tyr:NRasQ61K;Ink4a/Arf−/−;TR3+/+ and Tyr:NRasQ61K;Ink4a/Arf−/−;TR3−/− mice are shown (n > 16).


RNAi was induced with an empty vector as a negative control ( panel I ) , whereas a smg - 2 clone ( panel II ) was used as a positive control . Panels i and ii show brightfield images of the PTCxi strain treated with the negative and positive controls , respectively . Depletion of five genes ( panels III to VII ) resulted in increased GFP expression . Panels iii to vii show brightfield images of the phenotypes of the affected worms . The scale bars correspond to 100 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

RNAi was induced with an empty vector as a negative control (panel I), whereas a smg-2 clone (panel II) was used as a positive control. Panels i and ii show brightfield images of the PTCxi strain treated with the negative and positive controls, respectively. Depletion of five genes (panels III to VII) resulted in increased GFP expression. Panels iii to vii show brightfield images of the phenotypes of the affected worms. The scale bars correspond to 100 μm.


Depletion of the novel NMD genes in C . elegans leads to upregulation of the PTCx NMD reporter mRNA , which was monitored by quantitative RT - PCR relative to the expression of ama - 1 reference gene . The values shown are the average fold - change ( mean ± SEM ) from at least three independent experiments relative to empty vector - depleted worms . Statistical analysis was performed using the Mann - Whitney U - test for non - parametric distributions . * 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

Depletion of the novel NMD genes in C. elegans leads to upregulation of the PTCx NMD reporter mRNA, which was monitored by quantitative RT-PCR relative to the expression of ama-1 reference gene. The values shown are the average fold-change (mean ± SEM) from at least three independent experiments relative to empty vector-depleted worms. Statistical analysis was performed using the Mann-Whitney U-test for non-parametric distributions. *P < 0.05.


A - C Embryos expressing a GFP - NMD sensor ( green ) under the control of nompA regulatory sequences show signal in support cells linked to the embryonic peripheral nervous system ( PNS ) [ labeled by 22C10 signal ( magenta ) ] . DAPI signal is shown in blue . Expression of UAS - RNAi constructs against Upf1 ( B ) or nompA ( C ) genes using a nompA - Gal4 driver leads to upregulation of the GFP - NMD sensor when compared to wild - type ( no RNAi ) ( A ) , revealing a reduction in NMD activity in the knockdown conditions . Scale bars represent 100 μm . D - F Higher magnification ( 40 × ) of the areas marked by a rectangle in ( A - C ) further illustrates the upregulation of the GFP - NMD sensor in Upf1 - ( E ) and NompA - depleted cells ( F ) . Optical fields include embryonic abdominal segments A4 - A5 . Scale bars represent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Embryos expressing a GFP-NMD sensor (green) under the control of nompA regulatory sequences show signal in support cells linked to the embryonic peripheral nervous system (PNS) [labeled by 22C10 signal (magenta)]. DAPI signal is shown in blue. Expression of UAS-RNAi constructs against Upf1 (B) or nompA (C) genes using a nompA-Gal4 driver leads to upregulation of the GFP-NMD sensor when compared to wild-type (no RNAi) (A), revealing a reduction in NMD activity in the knockdown conditions. Scale bars represent 100 μm.D-F Higher magnification (40×) of the areas marked by a rectangle in (A-C) further illustrates the upregulation of the GFP-NMD sensor in Upf1- (E) and NompA-depleted cells (F). Optical fields include embryonic abdominal segments A4-A5. Scale bars represent 10 μm.


G Quantification of GFP signal in cells marked by an arrow in panels ( D - F ) shows a significant upregulation of NMD sensor expression upon downregulation of Upf1 ( dark gray ) and NompA ( black ) compared to wild - type ( no RNAi ) ( light gray ) . Results represent the average of five biological replicates ( mean ± SEM ) . Pair - wise comparisons were performed using a one - tailed t - test ( non - parametric ) between treatments and wild - type . * * * P < 0 . 001 .

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

G Quantification of GFP signal in cells marked by an arrow in panels (D-F) shows a significant upregulation of NMD sensor expression upon downregulation of Upf1 (dark gray) and NompA (black) compared to wild-type (no RNAi) (light gray). Results represent the average of five biological replicates (mean ± SEM). Pair-wise comparisons were performed using a one-tailed t-test (non-parametric) between treatments and wild-type. ***P < 0.001.


A , B HeLa cells stably expressing a wild - type β - globin reporter ( A ) or a β - globin NS39 NMD reporter ( B ) were mock - depleted or depleted of UPF2 , GNL2 , SEC13 , RAB27A , RAB27B or both RAB27A and RAB27B . The level of the β - globin mRNA was monitored by quantitative RT - PCR relative to two reference genes ( POLR2J and ACTB ) . The values shown are the average fold - change ( mean ± SEM ) from four independent experiments relative to mock - depleted cells ( control ) . Statistical analysis was performed using the Mann - Whitney U - test for non - parametric distributions . * P < 0 . 05 . The level of depletion of NMD factors is shown in Supplementary Fig S3C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B HeLa cells stably expressing a wild-type β-globin reporter (A) or a β-globin NS39 NMD reporter (B) were mock-depleted or depleted of UPF2, GNL2, SEC13, RAB27A, RAB27B or both RAB27A and RAB27B. The level of the β-globin mRNA was monitored by quantitative RT-PCR relative to two reference genes (POLR2J and ACTB). The values shown are the average fold-change (mean ± SEM) from four independent experiments relative to mock-depleted cells (control). Statistical analysis was performed using the Mann-Whitney U-test for non-parametric distributions. *P < 0.05. The level of depletion of NMD factors is shown in Supplementary Fig S3C.


C , D Analysis of the half - life of β - globin reporters . Samples were collected at the indicated time points , and the mRNA levels of the HBB reporters were monitored by qRT - PCR and normalized to POLR2J and ACTB reference genes . The values shown are the average fold - change ( mean ± SEM ) from three independent experiments relative to the first time point .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D Analysis of the half-life of β-globin reporters. Samples were collected at the indicated time points, and the mRNA levels of the HBB reporters were monitored by qRT-PCR and normalized to POLR2J and ACTB reference genes. The values shown are the average fold-change (mean ± SEM) from three independent experiments relative to the first time point.


E Depletion of GNL2 and SEC13 leads to a significant upregulation in the mRNA levels of endogenous NMD substrates . Samples were analyzed as described in ( A , B ) * P < 0 . 05 ; * * * P < 0 . 01 , * * * P < 0 . 001 .

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

E Depletion of GNL2 and SEC13 leads to a significant upregulation in the mRNA levels of endogenous NMD substrates. Samples were analyzed as described in (A, B) *P < 0.05; ***P < 0.01, ***P < 0.001.


F GNL2 and SEC13 contribute to the negative NMD feedback loop , regulating the levels of transcripts encoding NMD factors . RT - qPCR analysis of total cellular RNA from HeLa cells depleted of GNL2 ( in green ) and SEC13 ( in purple ) is shown . The graph shows distribution of relative fold - change from eight independent experiments relative to mock - depleted cells ( control ) . Statistical analysis was performed using Student ' s t - 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 O O O O O O

F GNL2 and SEC13 contribute to the negative NMD feedback loop, regulating the levels of transcripts encoding NMD factors. RT-qPCR analysis of total cellular RNA from HeLa cells depleted of GNL2 (in green) and SEC13 (in purple) is shown. The graph shows distribution of relative fold-change from eight independent experiments relative to mock-depleted cells (control). Statistical analysis was performed using Student's t-test. *P < 0.05.


C . Coomassie staining of SDS - PAGE - separated pulldown samples with / without cell extracts ( C . E ) using GST - tagged FET - NTDs as baits . Sepharose with bound GST was included as control . Background from recombinant protein baits ( shown by white arrowheads ) as well as smaller partial recombinant products is visualized in the left panel ( C . E - ) . Several high - molecular weight proteins are retained by the FET - NTDs ( C . E + ) . Black arrowheads indicate protein bands and gel parts analyzed by mass

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Coomassie staining of SDS-PAGE-separated pulldown samples with/without cell extracts (C.E) using GST-tagged FET-NTDs as baits. Sepharose with bound GST was included as control. Background from recombinant protein baits (shown by white arrowheads) as well as smaller partial recombinant products is visualized in the left panel (C.E -). Several high-molecular weight proteins are retained by the FET-NTDs (C.E +). Black arrowheads indicate protein bands and gel parts analyzed by mass spectrometry


D . Immunoblot analysis ( IB ) of pulldown samples with FET - NTD baits with / without cell extracts ( C . E ) . Antibodies against ARID1A , BRG1 and BAF60A were used for detection of SWI / SNF components . Input samples diluted 1 : 5 were included as a control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis (IB) of pulldown samples with FET-NTD baits with/without cell extracts (C.E). Antibodies against ARID1A, BRG1 and BAF60A were used for detection of SWI/SNF components. Input samples diluted 1:5 were included as a control.


E . In situ proximity ligation assays ( PLA ) using antibodies against BRG1 , ARID1A , DDIT3 and C - terminal parts of normal FUS show protein complexes containing FUS / BRG1 , FUS / ARID1A , FUS - DDIT3 / BRG1 and FUS - DDIT3 / ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645 - 94 and 402 - 91 . C - terminal parts of FUS are not present in the FUS - DDIT3 fusion protein and normal DDIT3 is not expressed in these cell lines . Merged images also include DAPI nuclear counterstain in blue . Combinations of primary antibodies are used to detect interactions ( left panel ) . In control experiments ( right panel ) , one primary antibody is omitted to evaluate the background fluorescent signals . Scale bars = 10 μm .

E. In situ proximity ligation assays (PLA) using antibodies against BRG1, ARID1A, DDIT3 and C-terminal parts of normal FUS show protein complexes containing FUS/BRG1, FUS/ARID1A, FUS-DDIT3/BRG1 and FUS-DDIT3/ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645-94 and 402-91. C-terminal parts of FUS are not present in the FUS-DDIT3 fusion protein and normal DDIT3 is not expressed in these cell lines. Merged images also include DAPI nuclear counterstain in blue. Combinations of primary antibodies are used to detect interactions (left panel). In control experiments (right panel), one primary antibody is omitted to evaluate the background fluorescent signals. Scale bars = 10 μm.


B . Immunoblot analysis ( IB ) of GST - pulldown samples with deletion mutants of FUS ‑ NTD ‑ GST as baits with / without cell extract ( C . E ) . ARID1A and BRG1 were used as tracers of SWI / SNF binding . Coomassie staining of recombinant proteins are shown in the lower panel . Amino acid numbers are indicated

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis (IB) of GST-pulldown samples with deletion mutants of FUS‑NTD‑GST as baits with/without cell extract (C.E). ARID1A and BRG1 were used as tracers of SWI/SNF binding. Coomassie staining of recombinant proteins are shown in the lower panel. Amino acid numbers are indicated


C . Immunoblot analysis ( IB ) of FUS - NTD ‑ GST pulldown samples of cell extracts ( C . E . ) after treatment with RNase or DNase , using antibodies against ARID1A and BRG1

O O O O O O O O O O O O O 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. Immunoblot analysis (IB) of FUS-NTD‑GST pulldown samples of cell extracts (C.E.) after treatment with RNase or DNase, using antibodies against ARID1A and BRG1


D . Immunoblot analysis ( IB ) of SWI / SNF components ( ARID1A , BRG1 , BAF170 , BAF155 , BAF60A , SS18 and BAF47 ) and full - length FET proteins ( EWSR1 and FUS ) remaining on sepharose - bound FUS - NTD ‑ GST pulldown baits after increasing stringency washes with 100 , 150 , 250 , 500 or 1000 mM NaCl . Note loss of BRG1 at NaCl concentration > 250mM

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

D. Immunoblot analysis (IB) of SWI/SNF components (ARID1A, BRG1, BAF170, BAF155, BAF60A, SS18 and BAF47) and full-length FET proteins (EWSR1 and FUS) remaining on sepharose-bound FUS-NTD‑GST pulldown baits after increasing stringency washes with 100, 150, 250, 500 or 1000 mM NaCl. Note loss of BRG1 at NaCl concentration > 250mM


A . Immunoblot analysis ( IB ) of proteins co - immunoprecipitated with nuclear extracted SWI / SNF from MLS cell line 402 ‑ 91 and EWS cell line TC - 71 . Antibodies against BRG1 , DDIT3 ( FUS - DDIT3 ) , the C - terminal parts of normal FUS and EWSR1 , and FLI1 ( EWSR1 - FLI1 ) were used for detection . In order to directly quantify the fraction of bound and non - bound protein , relative amounts of protein for each IP ‑ sample were loaded on the gel , with consideration taken for dilutions during the immunoprecipitation procedure . I : input of nuclear extract , B : bound proteins , NB : proteins not bound . One representative immunoblot is shown . Immunoblots from all replicates are shown in the source data . B . Graphs showing the percentage of bound to total ( bound + non ‑ bound ) signal intensities from immunoblots for FUS ‑ DDIT3 , EWSR1 and FUS in MLS 402 - 91 cell line and EWSR1 - FLI1 , EWSR1 and FUS in EWS TC - 71 cell line . Mean ± SEM is shown with individual replicates indicated by circles ( n = 3 ) . Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Original data for all quantifications , including p - values , are shown as source data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis (IB) of proteins co-immunoprecipitated with nuclear extracted SWI/SNF from MLS cell line 402‑91 and EWS cell line TC-71. Antibodies against BRG1, DDIT3 (FUS-DDIT3), the C-terminal parts of normal FUS and EWSR1, and FLI1 (EWSR1-FLI1) were used for detection. In order to directly quantify the fraction of bound and non-bound protein, relative amounts of protein for each IP‑sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. One representative immunoblot is shown. Immunoblots from all replicates are shown in the source data. B. Graphs showing the percentage of bound to total (bound + non‑bound) signal intensities from immunoblots for FUS‑DDIT3, EWSR1 and FUS in MLS 402-91 cell line and EWSR1-FLI1, EWSR1 and FUS in EWS TC-71 cell line. Mean ± SEM is shown with individual replicates indicated by circles (n=3). Student's t-test, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data.


C . Immunoblot analysis of proteins co - immunoprecipitated with nuclear extracted SWI / SNF from the model cell line HT1080 with overexpressed FUS - DDIT3 - dsRED , EWSR1 - FLI1 - EGFP or dsRED are shown as representative immunoblots . Antibodies against BRG1 , DDIT3 ( FUS - DDIT3 - dsRED ) , the C ‑ terminal parts of normal FUS and EWSR1 , EZH2 and FLI1 ( EWSR1 - FLI1 - EGFP ) were used for detection . In order to directly quantify the fraction of bound and non - bound protein , relative amounts of protein for each IP ‑ sample were loaded on the gel , with consideration taken for dilutions during the immunoprecipitation procedure . I : input of nuclear extract , B : bound proteins , NB : proteins not bound . Immunoblots from all replicates are shown in the source data ( with either dsRED - or EGFP ‑ tagged proteins expressed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis of proteins co-immunoprecipitated with nuclear extracted SWI/SNF from the model cell line HT1080 with overexpressed FUS-DDIT3-dsRED, EWSR1-FLI1-EGFP or dsRED are shown as representative immunoblots. Antibodies against BRG1, DDIT3 (FUS-DDIT3-dsRED), the C‑terminal parts of normal FUS and EWSR1, EZH2 and FLI1 (EWSR1-FLI1-EGFP) were used for detection. In order to directly quantify the fraction of bound and non-bound protein, relative amounts of protein for each IP‑sample were loaded on the gel, with consideration taken for dilutions during the immunoprecipitation procedure. I: input of nuclear extract, B: bound proteins, NB: proteins not bound. Immunoblots from all replicates are shown in the source data (with either dsRED- or EGFP‑tagged proteins expressed).


D . Graphs showing the percentage of bound to total ( bound + non ‑ bound ) signal intensities from immunoblots for the fusion protein ( FUS ‑ DDIT3 or EWSR1 - FLI1 ) , EWSR1 , FUS and EZH2 in the model cell line HT1080 with overexpressed FUS - DDIT3 - dsRED / - EGFP ( n = 4 , circles ) , EWSR1 - FLI1 - EGFP ( n = 3 , squares ) or dsRED / EGFP ( n = 4 , triangles ) . Mean ± SEM is shown with individual replicates indicated . Student ' s t - test , no significant changes , p > 0 . 05 . Original data for all quantifications , including p - values , are shown as source data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Graphs showing the percentage of bound to total (bound + non‑bound) signal intensities from immunoblots for the fusion protein (FUS‑DDIT3 or EWSR1-FLI1), EWSR1, FUS and EZH2 in the model cell line HT1080 with overexpressed FUS-DDIT3-dsRED/-EGFP (n=4, circles), EWSR1-FLI1-EGFP (n=3, squares) or dsRED/EGFP (n=4, triangles). Mean ± SEM is shown with individual replicates indicated. Student's t-test, no significant changes, p>0.05. Original data for all quantifications, including p-values, are shown as source data.


A . Immunoblot analysis ( IB ) of histone modifications in stably transfected HT1080 cell lines expressing EGFP , FUS ‑ DDIT3 ‑ EGFP or EWSR1 ‑ FLI1 ‑ EGFP . Antibodies against H3K27Ac , H3K27me3 , H3K4me3 and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount . Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP . One representative immunoblot is shown . Graphs showing amount of protein ( H3K27me3 , H3K4me3 , ratio H3K27me3 / H3K4me3 , H3K27Ac , and EZH2 ) quantified from immunoblots relative each corresponding loading control H4 or GAPDH , normalized to parental HT1080 . Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection and by squares from two experiments with transient transfection ( 24h and 48h , n = 4 . Student ' s t - test , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Original data for all quantifications , including p - values , are shown as source data .

O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis (IB) of histone modifications in stably transfected HT1080 cell lines expressing EGFP, FUS‑DDIT3‑EGFP or EWSR1‑FLI1‑EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3 and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. One representative immunoblot is shown. Graphs showing amount of protein (H3K27me3, H3K4me3, ratio H3K27me3/H3K4me3, H3K27Ac, and EZH2) quantified from immunoblots relative each corresponding loading control H4 or GAPDH, normalized to parental HT1080. Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection and by squares from two experiments with transient transfection (24h and 48h , n=4. Student's t-test, ns= not significant, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data.


A . HeLa cells released from double thymidine synchronization in early S phase were treated with 5 - azadC for 30 min , washed and collected at the indicated times . Soluble and chromatin - enriched fractions were immunoblotted with indicated antibodies .

O O O O O O O O O O O O O O O O O O O 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. HeLa cells released from double thymidine synchronization in early S phase were treated with 5-azadC for 30 min, washed and collected at the indicated times. Soluble and chromatin-enriched fractions were immunoblotted with indicated antibodies.


E . HeLa / GFP - DNMT1 cells transfected with indicated siRNAs were processed as in ( D ) .

E. HeLa/GFP-DNMT1 cells transfected with indicated siRNAs were processed as in (D).


H . Representative images of U2OS cells transfected with indicated siRNAs followed by mCherry - RNF4 expression plasmid . Cells were treated with 5 - azadC in the presence or absence of SUMOi , fixed 2 h later , pre - extracted and co - immunostained with DNMT1 and SUMO2 / 3 antibodies . Scale bar , 5 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

H. Representative images of U2OS cells transfected with indicated siRNAs followed by mCherry-RNF4 expression plasmid. Cells were treated with 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and SUMO2/3 antibodies. Scale bar, 5 μm.


J . GFP - tagged DNMT1 from extracts of HeLa / GFP - DNMT1 cells treated or not with 5 - azadC was immobilized on GFP - Trap agarose , subjected to stringent washing to remove proteins non - covalently bound to GFP - DNMT1 and incubated with recombinant HA - ubiquitin , E1 and E2 ( UbcH5a ) enzymes and RNF4 proteins ( STUbL reaction ) at 37 ° C for 1 h . Samples were then subjected to immunoblotting to assay for RNF4 - dependent STUbL activity towards GFP - DNMT1 .

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J. GFP-tagged DNMT1 from extracts of HeLa/GFP-DNMT1 cells treated or not with 5-azadC was immobilized on GFP-Trap agarose, subjected to stringent washing to remove proteins non-covalently bound to GFP-DNMT1 and incubated with recombinant HA-ubiquitin, E1 and E2 (UbcH5a) enzymes and RNF4 proteins (STUbL reaction) at 37 °C for 1 h. Samples were then subjected to immunoblotting to assay for RNF4-dependent STUbL activity towards GFP-DNMT1.


A . M . HpaII was crosslinked into a plasmid to generate p4xDPC and then incubated in nucleoplasmic egg extracts ( NPE ) in the presence or absence of 50 μM of SUMOi . DPC pull - down under stringent conditions was performed at the indicated time points and the recovered samples were immunoblotted for crosslinked M . HpaII ( Larsen et al , 2019 ) . Note that p4xDPC , which contains four M . HpaII DPCs , is sometimes used to increase sensitivity towards modified M . HpaII species . Identical results were obtained with plasmids containing one or four DPCs .

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A. M.HpaII was crosslinked into a plasmid to generate p4xDPC and then incubated in nucleoplasmic egg extracts (NPE) in the presence or absence of 50 μM of SUMOi. DPC pull-down under stringent conditions was performed at the indicated time points and the recovered samples were immunoblotted for crosslinked M.HpaII (Larsen et al, 2019). Note that p4xDPC, which contains four M.HpaII DPCs, is sometimes used to increase sensitivity towards modified M.HpaII species. Identical results were obtained with plasmids containing one or four DPCs.


B . p4xDPC or p4xDPCSUMO ( generated by crosslinking SUMO ∆ GG - M . HpaII ) were incubated in NPE and DPC pull - down performed as in ( A ) . Note that priming M . HpaII with SUMO stimulates rapid poly - SUMOylation of the DPC in NPE . Thus , this substrate was frequently used in subsequent experiments to stimulate DPC poly - SUMOylation .

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B. p4xDPC or p4xDPCSUMO (generated by crosslinking SUMO∆GG-M.HpaII) were incubated in NPE and DPC pull-down performed as in (A). Note that priming M.HpaII with SUMO stimulates rapid poly-SUMOylation of the DPC in NPE. Thus, this substrate was frequently used in subsequent experiments to stimulate DPC poly-SUMOylation.


C . p4xDPCSUMO was incubated in NPE and plasmids analyzed as in ( A ) . Following plasmid pull - down , the samples were split and either left untreated or treated with the SUMO protease Ulp1 .

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C. p4xDPCSUMO was incubated in NPE and plasmids analyzed as in (A). Following plasmid pull-down, the samples were split and either left untreated or treated with the SUMO protease Ulp1.


I . Representative images of U2OS cells transfected with Myc - PIAS4 expression plasmid that were left untreated or exposed to 5 - azadC in the presence or absence of SUMOi , fixed 2 h later , pre - extracted and co - immunostained with DNMT1 and Myc antibodies . Scale bar , 5 μm .

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I. Representative images of U2OS cells transfected with Myc-PIAS4 expression plasmid that were left untreated or exposed to 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and Myc antibodies. Scale bar, 5 μm.


J . HeLa or HeLa / GFP - DNMT1 cells left untreated or exposed to 5 - azadC for 30 min were lysed and subjected to GFP IP under stringent conditions . After extensive washing , individual IPs were incubated with an equal amount ( 800 μg ) of whole cell lysate of HeLa cells transfected with Myc - PIAS4 expression construct ( Fig . EV2J ) , washed and immunoblotted with antibodies to Myc , SUMO2 / 3 and GFP . * , cross - reactive band .

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J. HeLa or HeLa/GFP-DNMT1 cells left untreated or exposed to 5-azadC for 30 min were lysed and subjected to GFP IP under stringent conditions. After extensive washing, individual IPs were incubated with an equal amount (800 μg) of whole cell lysate of HeLa cells transfected with Myc-PIAS4 expression construct (Fig. EV2J), washed and immunoblotted with antibodies to Myc, SUMO2/3 and GFP. *, cross-reactive band.


K . HeLa / GFP - DNMT1 cells transfected with previously validated siRNAs targeting established SUMO E3 ligases ( Fig . EV2L and Methods section ) were treated with 5 - azadC for 30 min , collected and subjected to GFP immunoprecipitation under denaturing conditions , and immunoblotted with antibodies to SUMO1 , SUMO2 / 3 , ubiquitin and GFP .

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K. HeLa/GFP-DNMT1 cells transfected with previously validated siRNAs targeting established SUMO E3 ligases (Fig. EV2L and Methods section) were treated with 5-azadC for 30 min, collected and subjected to GFP immunoprecipitation under denaturing conditions, and immunoblotted with antibodies to SUMO1, SUMO2/3, ubiquitin and GFP.


B . p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [ α - 32P ] dATP . Where indicated , 50 μM of SUMOi was added to the extracts . Reaction samples were analyzed by native agarose gel electrophoresis . RI , replication intermediates ; OC , open circular ; SC , supercoiled . Red arrowheads indicate OC molecules that have not yet undergone repair . C . Samples in ( B ) were recovered by stringent DPC pull - down and immunoblotted for crosslinked M . HpaII .

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B. p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [α-32P]dATP. Where indicated, 50 μM of SUMOi was added to the extracts. Reaction samples were analyzed by native agarose gel electrophoresis. RI, replication intermediates; OC, open circular; SC, supercoiled. Red arrowheads indicate OC molecules that have not yet undergone repair. C. Samples in (B) were recovered by stringent DPC pull-down and immunoblotted for crosslinked M.HpaII.


B . p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [ α - 32P ] dATP . Where indicated , 50 μM of SUMOi was added to the extracts . Reaction samples were analyzed by native agarose gel electrophoresis . RI , replication intermediates ; OC , open circular ; SC , supercoiled . Red arrowheads indicate OC molecules that have not yet undergone repair .

B. p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [α-32P]dATP. Where indicated, 50 μM of SUMOi was added to the extracts. Reaction samples were analyzed by native agarose gel electrophoresis. RI, replication intermediates; OC, open circular; SC, supercoiled. Red arrowheads indicate OC molecules that have not yet undergone repair.


J . As in ( I ) , but using RNF4 - depleted CSF - arrested extracts reconstituted with recombinant RNF4 WT or C130A from ( H ) .

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J. As in (I), but using RNF4-depleted CSF-arrested extracts reconstituted with recombinant RNF4 WT or C130A from (H).


L . p4xDPCSUMO was subjected to sequential addition of NPE and CSF - arrested extract as in ( I ) . CSF - arrested extracts from ( K ) were supplemented with recombinant RNF4 , and the DPC plasmid was recovered by DPC pull - down at the indicated time points and immunoblotted against M . HpaII .

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L. p4xDPCSUMO was subjected to sequential addition of NPE and CSF-arrested extract as in (I). CSF-arrested extracts from (K) were supplemented with recombinant RNF4, and the DPC plasmid was recovered by DPC pull-down at the indicated time points and immunoblotted against M.HpaII.


D . HeLa cells transfected with indicated siRNAs and synchronized in early S phase by double thymidine block were pulse - labeled with 5 - azadC in late S phase as outlined in ( A ) . Cells were then collected at the indicated times after 5 - azadC withdrawal and analyzed by flow cytometry . Data are representative of three independent experiments . Proportion of cells with G2 / M DNA content is indicated .

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D. HeLa cells transfected with indicated siRNAs and synchronized in early S phase by double thymidine block were pulse-labeled with 5-azadC in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.


E . HeLa cells transfected with indicated siRNAs were treated with 5 - azadC and / or SUMOi in late S phase as outlined in ( A ) . Cells were then collected at the indicated times after 5 - azadC withdrawal and analyzed by flow cytometry . Data are representative of three independent experiments . Proportion of cells with G2 / M DNA content is indicated .

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E. HeLa cells transfected with indicated siRNAs were treated with 5-azadC and/or SUMOi in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.


F . HeLa cells transfected with indicated siRNAs were pulse - labeled or not with 5 - azadC for 30 min in late S phase according to the experimental setup in ( A ) . Following 5 - azadC withdrawal cells were incubated with nocodazole , collected at the indicated times and immunoblotted with indicated antibodies .

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F. HeLa cells transfected with indicated siRNAs were pulse-labeled or not with 5-azadC for 30 min in late S phase according to the experimental setup in (A). Following 5-azadC withdrawal cells were incubated with nocodazole, collected at the indicated times and immunoblotted with indicated antibodies.


H . Immunoblot analysis of siRNA - treated HeLa cells that were exposed to indicated genotoxic agents and collected 1 h later .

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H. Immunoblot analysis of siRNA-treated HeLa cells that were exposed to indicated genotoxic agents and collected 1 h later.


B . Representative time - lapse sequences of mitotic progression in cells in ( A ) . NEBD corresponds to t = 0 . White arrowheads indicate cells undergoing division . Scale bar , 10 μm .

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B. Representative time-lapse sequences of mitotic progression in cells in (A). NEBD corresponds to t=0. White arrowheads indicate cells undergoing division. Scale bar, 10 μm.


E . HeLa cells transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block . Six h after release from the block , cells were pulse - labeled with 5 - azadC for 30 min . Mitotic cells were isolated by shake - off 8 h later , washed and incubated or not with MPS1 inhibitor ( MPS1i ) and collected at the indicated times . Cells were then processed for immunoblotting with indicated antibodies .

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E. HeLa cells transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block. Six h after release from the block, cells were pulse-labeled with 5-azadC for 30 min. Mitotic cells were isolated by shake-off 8 h later, washed and incubated or not with MPS1 inhibitor (MPS1i) and collected at the indicated times. Cells were then processed for immunoblotting with indicated antibodies.


A - B Proteins of GS and GS2KR ( A ) and GDH and GDHK128R ( B ) were expressed from Salmonella in LB in the presence or absence of 50 mM NAM . : The acetylation levels of GS and GDH were assayed by immunoblotting . Quantitation of signals is shown . Immunoblots represent at least two independent experiments For all immunoblotting data , molecular weights in kD are indicated to the right .

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A-B Proteins of GS and GS2KR (A) and GDH and GDHK128R (B) were expressed from Salmonella in LB in the presence or absence of 50 mM NAM. : The acetylation levels of GS and GDH were assayed by immunoblotting. Quantitation of signals is shown. Immunoblots represent at least two independent experiments For all immunoblotting data, molecular weights in kD are indicated to the right.


GS ( C ) were expressed from Salmonella in minimal media with 50 mM glucose and various concentrations of nitrate ( left ) and ammonium ( right ) , as indicated . The acetylation levels of GS were assayed by immunoblotting . Quantitation of signals is shown . Immunoblots represent at least two independent experiments For all immunoblotting data , molecular weights in kD are indicated to the right .

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GS (C) were expressed from Salmonella in minimal media with 50 mM glucose and various concentrations of nitrate (left) and ammonium (right), as indicated. The acetylation levels of GS were assayed by immunoblotting. Quantitation of signals is shown. Immunoblots represent at least two independent experiments For all immunoblotting data, molecular weights in kD are indicated to the right.


E - F GS ( E ) or GDH ( F ) were expressed from Salmonella under LB and minimal media with 50 mM glucose , 50 mM 2 - DG , and 25 mM lactose as indicated . : The acetylation levels of GS and GDH were assayed by immunoblotting . Quantitation of signals is shown . Immunoblots represent at least two independent experiments For all immunoblotting data , molecular weights in kD are indicated to the right .

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E-F GS (E) or GDH (F) were expressed from Salmonella under LB and minimal media with 50 mM glucose, 50 mM 2-DG, and 25 mM lactose as indicated. : The acetylation levels of GS and GDH were assayed by immunoblotting. Quantitation of signals is shown. Immunoblots represent at least two independent experiments For all immunoblotting data, molecular weights in kD are indicated to the right.


A - B GS ( A ) or GDH ( B ) were expressed from Salmonella in minimal media with 100 mM ammonium and various glucose concentrations as indicated . The specific activity of GS and GDH from 2 mM glucose was set as a control for statistical analysis . : Acetylation levels were determined by immunoblotting . The specific activities of GS and GDH were measured spectrophotometrically and compared . The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity . Immunoblots represent at least two independent

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A-B GS (A) or GDH (B) were expressed from Salmonella in minimal media with 100 mM ammonium and various glucose concentrations as indicated. The specific activity of GS and GDH from 2 mM glucose was set as a control for statistical analysis. : Acetylation levels were determined by immunoblotting. The specific activities of GS and GDH were measured spectrophotometrically and compared. The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Immunoblots represent at least two independent experiments


C In vivo GS - specific activity of Salmonella cultured in LB with or without NAM . The specific activity of GS from cells untreated by NAM was set as the statistical control . : Acetylation levels were determined by immunoblotting . The specific activities of GS were measured spectrophotometrically and compared . The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity . Immunoblots represent at least two independent

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C In vivo GS-specific activity of Salmonella cultured in LB with or without NAM. The specific activity of GS from cells untreated by NAM was set as the statistical control. : Acetylation levels were determined by immunoblotting. The specific activities of GS were measured spectrophotometrically and compared. The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Immunoblots represent at least two independent experiments


GSs ( F ) were expressed and purified from Salmonella cultured in minimal media with high ( 100 mM ) or low ( 2 mM ) ammonium supplemented with high ( 50 mM ) or low ( 2 mM ) glucose . The specific activity of GS from 2 mM glucose was set as a control for statistical analysis . Acetylation levels were determined by immunoblotting . The specific activities of GS were measured spectrophotometrically and compared . The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity . Immunoblots represent at least two independent experiments Each symbol represents an independent biological replicate Data are from three independent biological replicates and expressed as the mean ±

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GSs (F) were expressed and purified from Salmonella cultured in minimal media with high (100 mM) or low (2 mM) ammonium supplemented with high (50 mM) or low (2 mM) glucose. The specific activity of GS from 2 mM glucose was set as a control for statistical analysis. Acetylation levels were determined by immunoblotting. The specific activities of GS were measured spectrophotometrically and compared. The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Immunoblots represent at least two independent experiments Each symbol represents an independent biological replicate Data are from three independent biological replicates and expressed as the mean ± SD


H GDHWT , GDHK128Q and GDHK128R were expressed and purified from Salmonella in LB . The specific activity of GDHWT was set as a statistical reference .

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H GDHWT, GDHK128Q and GDHK128R were expressed and purified from Salmonella in LB. The specific activity of GDHWT was set as a statistical reference.