STL-based analysis of TRAIL-induced apoptosis challenges the notion of type I/type II cell line classification

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https://hal.inria.fr/hal-00750063

Submitted on 14 Nov 2012

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STL-based analysis of TRAIL-induced apoptosis

challenges the notion of type I/type II cell line

classification

Szymon Stoma, Alexandre Donzé, François Bertaux, Oded Maler, Grégory

Batt

To cite this version:

Szymon Stoma, Alexandre Donzé, François Bertaux, Oded Maler, Grégory Batt. STL-based analysis of TRAIL-induced apoptosis challenges the notion of type I/type II cell line classification. [Research Report] RR-8121, INRIA. 2012, pp.35. �hal-00750063�

STL-based analysis of TRAIL-induced

apoptosis challenges the notion of

type I/type II cell line classification

Szymon Stoma, Alexandre Donzé, François Bertaux, Oded Maler, Gregory Batt

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STL-based analysis of TRAIL-induced apoptosis challenges the notion of type I/type II cell line classification

Szymon Stoma1,#, Alexandre Donzé2,#,$, François Bertaux1, Oded Maler2 and Gregory Batt1

1

INRIA Paris-Rocquencourt, 78153 Le Chesnay Cedex, France

2

VERIMAG, CNRS and the University of Grenoble, Gières, France

Research Report

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—35 pages.

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Une étude de l’apoptose induite par TRAIL basée sur STL questionne la pertinence d’une

classification des lignées cellulaires en types I ou II

# # # # # # #

/0)120.#L’apoptose extrinsèque est un processus de mort cellulaire programmée qui est activé par la présence de ligands

extracellulaires, tels que TRAIL (TNF-related apoptosis inducing ligand). Selon la lignée cellulaire, les mécanismes spécifiques qui mènent à la mort cellulaire diffèrent. La caractérisation de ces différences est essentielle pour comprendre et pouvoir exploiter à des fins thérapeutiques la réponse apoptotique des cellules. Plusieurs aspects de l’apoptose diffèrent d’une lignée cellulaire à l’autre dont notamment /(i)/ l’ordre relatif de l’activation des caspases, /(ii)/ la nécessité de la MOMP (mitochondria outer membrane permeabilization) pour l’activation des caspases effectrices, et /(iii)/ la survie des lignées cellulaires qui surexpriment Bcl2, ce qui a conduit à classifier les lignées cellulaires en deux groupes, dits type I et type II.

Dans ce travail, nous discutons cette classification en type I ou II. Nous encodons les trois comportements distinctifs mentionnés plus haut dans un langage formel, appelé STL (signal temporal logic), et testons la validité d’un modèle précédemment publié de l’apoptose induite par TRAIL par rapport à des observations expérimentales réalisées sur différentes lignées cellulaires. Puis, nous utilisons de la recherche de paramètres guidé par les propriétés de STL pour résoudre quelques incompatibilités détectées entre modèle et données. Nous montrons que ces trois critères différents ne définissent pas une classification cohérente des lignées cellulaires en type I ou II, et suggérons des mutants susceptibles de présenter des comportements ambivalents. En particulier, ces résultats soulignent l’importance d’une boucle de rétroaction entre caspases, et réconcilie deux visions apparemment contradictoires sur l’importance des processus amonts ou avals pour la détermination du type cellulaire. Plus généralement, notre travail suggère que plutôt que d’être considérés comme des critères de définition pour la classification des lignées cellulaire en deux types, ces comportement distinctifs devraient être simplement regardés comme des caractéristiques de type I ou II.

Du point de vue méthodologique, ce travail illustre la pertinence des diagrammes STL, des données de population STL, et de la recherché de paramètres guidés par des formules en STL. De tels outils sont bien adaptés à l’acquisition de plus en plus rapide de connaissances hétérogènes sur des voies de transduction du signal complexes.

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XIAP Caspase − 3 HCT116 SKW6.4 T47D 1 2 3 4 5 x 104 1 2 3 4 5 6 7 8 9 x 105 −0.4 −0.3 −0.2 −0.1 0 0.1 0.2 0.3 0.4

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H HX S T

−0.5 0 0.5

Distribution of in silico predicted property 1 values

H HX S T

0 50 100

Percentage of property 1 satisfaction in cell population

H HX S T

False True

Expected vs in silico predicted property 1 satisfaction

HB HBX SB TB −0.5

0 0.5

Distribution of in silico predicted property 1 values

HB HBX SB TB 0

50 100

Percentage of property 1 satisfaction in cell population HB HBX SB TB False

True

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*+

:+5;8+*<#D.#EFE:#;+8-868)#-,);,)8AD#,-*7>,*754#74#=<;8#33#-899)#

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`GH* J+)(D* 78.'* &''4B67./+* /+(* '8/4,)* /%'(DE(* 78&7* .+* 78(* 0.D'7* 5&'(* UWUX* 0/,,/R'* (00(57/D* 5&'6&'('* &57.E&7./+A* &+)* .+* 78(* '(5/+)*5&'(A*78&7*UWUX*6D(5()('*(00(57/D*5&'6&'('*&57.E&7./+H*"8.'*Q4('7./+*8&'*%((+*).D(57,-*.+E('7.9&7()*.+* 2,)D.)9(* (7* &,H* I@a??L* %-* '7&.+.+9* 5(,,'* R.78* &+7.$5-7/58D/B(* 5* &+)* &+7.$5X21X* &+7.%/).('H* "8(* &478/D'* )(B/+'7D&7(*78&7*B/'7*/0*[g"??^*5(,,'*'8/R.+9*(00(57/D*5&'6&'('*&57.E&7./+*&,'/*'8/R*5-7/6,&'B.5*5-7/58D/B(* 5*,/5&,.V&7./+H*!7&7()*).00(D(+7,-A*5&'6&'($S*.'*+/7*&57.E(*4+7.,*UWUX*8&66(+'H*"8.'*.'*+/7*&,R&-'*7D4(*0/D*hc32X* [g"??^*5(,,'A*/D*0/D*WM$P5,@*hc32X*[g"??^*5(,,'e*&*'.9+.0.5&+7*6D/6/D7./+*ID('6(57.E(,-*@ai*&+)*>ai*/0*78(*5(,,'L* '8/R'* (00(57/D* 5&'6&'(* &57.E&7./+* .+* &%'(+5(* /0* 5-7/6,&'B.5* 5-7/58D/B(* 5* IO.94D(* S* /0* F;GLH* "8(* '&B(* (N6(D.B(+7* R&'* B&)(* 0/D* "]_Y* &+)* WM$P5,@* "]_Y* 5(,,'A* '8/R.+9* 78&7* 78('(* 5(,,'* %(8&E(* ,.Z(* [g"??^* 5(,,'e* 5&'6&'($S*.'*+/7*&57.E(*4+7.,*UWUX*8&66(+'*IO.94D(*_*/0*F;GLH*

"/* 7('7* 78(* 5/+'.'7(+5-* /0* M21U?H]* R.78* 78('(* /%'(DE&7./+'A* R(* (N6D(''* .+* !"#* 78(* 6D/6(D7-A* 7-6.5&,* /0* 7-6(* 33* %(8&E./D'A*78&7*5(,,'*D(B&.+*&,.E(*4+7.,*UWUX*8&66(+'H*\(*'.B6,-*RD.7(**6/7%6.8G&'(KLK*(6%"%!;%(!"#$%M(with !"#$%&'()*+,*-*+,*./.!"0123 as in Property 1. The 6%"%!;% operator states that the second property (!"#$%) must hold until the first property holds for the first time (KLK*). "8(* /554DD(+5(* /0* UWUX* .'* )(7(57()* %-* 78(* 7.7D&7./+*/0*26&0$?*%-*78(*D(,(&'()*5-7/58D/B(*5*7/*0/DB*78(*&6/67/'/B(H**\(*'&-*78&7*UWUX*8&66(+()*R8(+* B/D(* 78&+* ;ai* /0* 26&0$?* .'* %/4+)* 7/* 5-7/58D/B(* 5e* *KLK*&'( +7!NN6%%-+7!N./.!"0123( !"##$ %&'()#$ *+$ ,-)$ .&"/(""&-0$-,$12)#"2-3.4H*"8(+A*R(*4'()*PD(&58*7/*5/B647(*78(*!"#*).&9D&B*&''/5.&7()*R.78*XD/6(D7-*S*R.78* D('6(57*7/*[g"??^*5(,,'A*&+)*78(*XD/6(D7-*S*6/64,&7./+*)&7&*IO.94D(*^LH*W+(*'8/4,)*+/7(*78&7*,.Z(*.+*78(*!"+8!#$.* '(746* /0* 2,)D.)9(* &+)* 5/,,(&94('* I@a??LA* /+,-* 5(,,'* .+* R8.58* UWUX* 8&66(+()* R(D(* 7&Z(+* .+7/* &55/4+7e* R(* (N5,4)()*'4DE.E.+9*5(,,'*7/*5/B647(*'7&7.'7.5'H* XIAP caspase − 3 HCT116 SKW6.4 T47D 1 2 3 4 5 x 104 1 2 3 4 5 6 7 8 9 x 105 −10 −8 −6 −4 −2 0 2 4 6 8 x 10−3 H HX HB HBX S SB −0.02 −0.01 0 0.01

Distribution of in silico predicted property 2 values

H HX HB HBX S SB 0

50 100

Percentage of property 2 satisfaction in cell population H HX HB HBX S SB False

?]** !V-B/+*!7/B&A*2,(N&+)D(*Y/+V(A*OD&+5/.'*P(D7&4NA*W)()*U&,(DA*dD(9/D-*P&77* *

*

!"#$%&' G)' D%2,&%:>9C' ,=;B&' /";#%;+' ;3/' B:;:"B:"<B)'"8(* E&,4(* /0* XD/6(D7-* S* I*F:;+ HIHJ+ $'6'2%'+ 26!8'K+ *F:;+ L*277$''ML*27#.#26NDOP+ $'6'2%'+

@&JLQJMJLQJ#.#26N+ DOPGL* .'* D(6D('(+7()* &'* &* 04+57./+* /0* 78(* c32X* &+)* 5&'6&'($S* .+.7.&,* 5/+5(+7D&7./+'H* 2'* .+* O.94D(* SA* 5(,,* ,.+('* &D(*

6/'.7./+()*.+*78.'*).&9D&B*&55/D).+9*7/*78(.D*6D/7(.+*.+.7.&,*5/+5(+7D&7./+'H*W+(*5&+*'((*/+*78.'*).&9D&B*78&7*[g"??^*)(6,(7()*0D/B*&,,* c32X*IFc32XGjaL*&D(*6D().57()*+/7*7/*'&7.'0-*XD/6(D7-*SA*.+*5/+7D&).57./+*7/*(N6(D.B(+7&,*/%'(DE&7./+'H*"8(*B.+.B&,*c32X*5/+5(+7D&7./+*0/D* R8.58*&*,&D9(*6D/6/D7./+*/0*[g"??^*5(,,'*'7.,,*'&7.'0-*XD/6(D7-*S*.'*&D/4+)*?a]*B/,(54,('A*5/DD('6/+).+9*7/*78(*.+&57.E&7./+*/0*&7*B/'7*7R/* 78.D)'*/0*c32X*&57.E.7-*.+*hc32X*[g"??^*5(,,'H*I1.987L*Y.'7D.%47./+'*/0*78(*E&,4('*/0*XD/6(D7-*S*&5D/''*6/64,&7./+'*/0*5(,,'*/0*).00(D(+7*5(,,* ,.+('H*</7&7./+'*&D(*.)(+7.5&,*7/*78/'(*4'()*.+*O.94D(*]H*W+(*5&+*+/7(*78&7*).'5D(6&+5.('*%(7R((+*6D().57()*B(&+*E&,4('*&+)*/%'(DE()* 68(+/7-6('*(N.'7*0/D*hc32X*[g"??^A*"]_Y*&+)*WM$P5,@*"]_Y*5(,,'H+ **

"8(* ).&9D&B* 6D('(+7()* .+* O.94D(* ^* .'* 5/+'.'7(+7* R.78* 78(* /%'(DE&7./+* 78&7* [g"??^* 5(,,'* '&7.'0-* 78(* 6D/6(D7-H* [/R(E(DA*.7*'499('7'*78&7*78(*hc32X*B47&+7*0&,'.0-*78(*6D/6(D7-A*'.+5(*+(9&7.E(*E&,4('*&D(*0/4+)*.+*78(*D(9./+* R8(D(* c32X* 5/+5(+7D&7./+* .'* +4,,H* "8.'* .'* .+* 5/+7D&).57./+* R.78* 78(* /%'(DE&7./+* 78&7* 5&'6&'($S* .'* &57.E(* %(0/D(* UWUX*8&66(+()*.+*&*B&C/D.7-*I>aiL*/0*78('(*5(,,'H*2,'/*5/+7D&).57./+'*&D(*0/4+)*R.78*"]_YA*&+)*WM$P5,@*"]_Y* 5(,,'* IO.94D(* ^A* D.987LH* "8(* 0&57* 78&7* hc32X* [g"??^* 5(,,'* 8&E(* %((+* /%'(DE()* 7/* '&7.'0-* XD/6(D7-* S* %47* +/7* XD/6(D7-* @* .B6/'('* '7D/+9* 5/+'7D&.+7'* /+* 78(* Z.+(7.5'* /0* 78(* &6/67/'.'* 6D/5(''H* 3+* 78('(* 5(,,'A* 5&'6&'($S* &57.E&7./+*.'*6D(5/5./4'A*'.+5(*.7*0/,,/R'*%-*,(''*78&+*/+(*8/4D*78(*&57.E&7./+*/0*5&'6&'($>A*.B6,-.+9*78&7*)(&78* I.H(H*X21X*5,(&E&9(L*.'*D&6.)*'.+5(*.7*0/,,/R'*'8/D7,-*&07(D*5&'6&'($S*&57.E&7./+H*P47*78(+*XD/6(D7-*S*.B6,.('*78&7* UWUX*8&66(+()*(E(+*%(0/D(*78.'*7.B(*.+'7&+7H*d.E(+*78(*(00.5.(+7*5&'6&'($S*&57.E&7./+*.+*M21U?H]A*78(*B/)(,* 0&.,'*7/*5&674D(*78(*+(()*0/D*UWUX*.+*78('(*5(,,'H*2,78/498*hc32X*[g"??^*5(,,'*6D('(+7*&*7-6(*3*68(+/7-6(*R.78* D('6(57* 7/* 5,/+/9(+.5* '4DE.E&,* &+)* 5&'6&'('* &57.E&7./+* )-+&B.5'A* &+)* &* 7-6(* 33* 68(+/7-6(* R.78* D('6(57* 7/* 78(* +(()*0/D*UWUX*0/D*5(,,*)(&78A*78(*B/)(,*5/+'.'7(+7,-*5,&''.0.('*78('(*5(,,'*&'*7-6(*3*5(,,'H*

*

32;+5>74G#H'/E#%IJ.#:+5;8+*<AG17686#;,+,28*8+#)8,+-K#

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caspase − 3 HCT116 SKW6.4 T47D 1 2 3 4 5 x 104 1 2 3 4 5 6 7 8 9 x 105 −0.4 −0.3 −0.2 −0.1 0 0.1 0.2 0.3 0.4 H HX HBX T TB −0.5 0 0.5

Distribution of in silico predicted property 3 values

H HX HBX T TB 0

50 100

Percentage of property 3 satisfaction in cell population H HX HBX T TB False

!"#$%&'()*&+&,-'.'*/0*"123#$.+)45()*&6/67/'.'*58&,,(+9('*78(*+/7./+*/0*7-6(*3:7-6(*33*5(,,*,.+(*5,&''.0.5&7./+*** ?;* *

'

H&--'-"3&'I'D%2,&%:>' D%2,&%:>'(' D%2,&%:>'?' D%2,&%:>'C'

JH4((G' O&,'(' "D4(' "D4('

KL76D'JH4((G'' O&,'(' O&,'(' 4%$&'

MA9N<-?'JH4((G' "D4(' "D4(' <:2'

MA9N<-?'KL76D'JH4((G' O&,'(' O&,'(' O&,'('

*OPG)E' O&,'(' !;-B&' <:2*'

MA9N<-?'*OPG)E' O&,'(' !;-B&' <:2'

4EQR' O&,'(' <:2' 4%$&'

MA9N<-?'4EQR' U.N()' <:2' 4%$&' '

4;S-&' (@' *$++;%>' 2.' ."3/"3#B)'"D478* E&,4('* /0* 78(* 78D((* 6D/6(D7.('* &'* /%'(DE()* .+* 2,)D.)9(* (7* &,H* I@a??L* 0/D* 78(* [g"??^A* !f\^H]A* &+)* "]_Y* 5(,,* ,.+('* &+)* '/B(* B47&+7'H* <:2* .+).5&7('* 78&7* 78(* (N6(D.B(+7&,* .+0/DB&7./+* .'* +/7* &E&.,&%,(H* MN6(D.B(+7'* '8/R()* 78&7* WM$P5,@* "]_Y*5(,,'*6D('(+7*5,/+/9(+.5*'4DE.E&,*D&7('*5,/'(*7/*;aiA*8(+5(*78(.D*kB.N()l*%(8&E./DH*Y.'5D(6&+5.('*/%7&.+()*R.78*78(*/D.9.+&,*M21U*

?H]* B/)(,* &D(* 8.98,.987()* .+* %/,)H*79&'(!":!8;<1=>?@4!"#$%5O(6@ej*%$%A.B!""84D!;7E!).#$%(!AF(!":!8;<1=9?@(A/.(D!;7G!).#$%5O(7G&'(KLK*(

6%"%!;%(!"#$%H*P(5&4'(*/0*78(.D*&B%.94/4'*68(+/7-6('A*"]_Y*5(,,*)&7&*I.+*9D(-L*R(D(*+/7*4'()*0/D*6&D&B(7(D*'(&D58H(

\(*0.D'7*7D.()*7/*D('/,E(*.+5/+'.'7(+5.('*%-*B.+/D*&)C4'7B(+7'*/0*78(*78D('8/,)'*R(*4'()*.+*0/DB4,&(H*[/R(E(DA* 6D/6(D7-*'&7.'0&57./+*E&,4('*6D/E()*D/%4'7*7/*78D('8/,)*58&+9('*IO.94D(*!;LH*\(*78(D(0/D(*D('/D7()*7/*'(&D58*0/D* %(77(D*6&D&B(7(D*E&,4('*4'.+9*9,/%&,*/67.B.V&7./+*B(78/)'*F;aGH*\(*)(0.+()*&*5/'7*04+57./+*78&7*.+).5&7('*0/D* &+-* 9.E(+* 6&D&B(7(D* 8/R* 0&D* 78(* B/)(,* .'* 0D/B* '&7.'0-.+9* &,,* .7'* 5/+'7D&.+7'H* U/D(* 6D(5.'(,-A* 78(* 5/'7* 04+57./+* &99D(9&7()*78D((*B(&'4D('e*8/R*B&+-*6D/6(D7.('*&D(*5/+'.'7(+7*R.78*78(*/%'(DE&7./+'A*8/R*D/%4'7,-*'&7.'0.()* /D*0&,'.0.()*78(-*&D(A*&+)*8/R*,&D9(*&D(*78(*)(E.&7./+'*/0*78(*6&D&B(7(D'*R.78*D('6(57*7/*78(.D*D(0(D(+5(*E&,4('H* "8(+A*R(*4'()*78(*9,/%&,*/67.B.V&7./+*7//,*gU2$M!*F;?G*7/*'(&D58*&47/B&7.5&,,-*0/D*6&D&B(7(D'*B.+.B.V.+9*78.'* 5/'7*04+57./+H*"8(*6&D&B(7(D*'(&D58*6D/5()4D(*.'*)('5D.%()*.+*)(7&.,'*.+*78(*U(78/)*'(57./+H*[(D(A*/+(*'8/4,)* +/7(*78&7*78(*D(&,$E&,4()*'(B&+7.5'*/0*!"#*6D/6(D7.('*.'*5D.7.5&,e*5/+7.+4/4'*/67.B.V&7./+*7//,'*7&Z(*&)E&+7&9(*/0* 78(*9D&)()*.+7(D6D(7&7./+*/0*!"#*6D/6(D7.('A*R8/'(*E&,4('*.+).5&7(*78(.D*k).'7&+5('*0D/B*'&7.'0&57./+l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d.E(+*78(*4'4&,,-*,&D9(*4+5(D7&.+7.('*.+*&574&,*6&D&B(7(D*E&,4('A*'458* 58&+9('* 5&+* '7.,,* %(* 5/+'.)(D()* &'* &55(67&%,(H* <(R* 6&D&B(7(D* E&,4('* ,(&)* 7/* '&7.'0&57./+* /0* XD/6(D7-* ?$S* .+*

".4!"26*5(,,'*5/DD('6/+).+9*7/*&,,*[g"??^*&+)*!f\^H]*+/DB&,*&+)*)(D.E()*5(,,*,.+('H*"/*7('7*R8(78(D*6D/6(D7-*

E&,4('*&D(*5/DD(57()*&7*78(*&'66+*.*162#!."*,(E(,A*R(*D(5/B647()*78(*6/64,&7./+*)&7&*R.78*78('(*+(R*6&D&B(7(D* E&,4('H* 2'* '8/R+* .+* O.94D(* _A* &,,* .+5/+'.'7(+5.('* R(D(* .+)(()* D('/,E()* &7* 78(* 5(,,* 6/64,&7./+* ,(E(,* 0/D* &,,* 5(,,* 7-6('*I&9&.+*R.78*78(*(N5(67./+*/0*"]_Y*5(,,'LH*\(*+/7(*78&7*"]_Y*5(,,'*'7.,,*)/*+/7*'&7.'0-*XD/6(D7-*S*IO.94D(*_* &+)*O.94D(*!;LH*

?^** !V-B/+*!7/B&A*2,(N&+)D(*Y/+V(A*OD&+5/.'*P(D7&4NA*W)()*U&,(DA*dD(9/D-*P&77* * * ' !"#$%&'Q@'D2,$-;:"23'B:;:"B:"<B'.2%'$,/;:&/'A65T()E'+2/&-)'X/64,&7./+*'7&7.'7.5'*0/D*XD/6(D7-*?A*@*&+)*SA*5/B647()*R.78*+(R*6&D&B(7(D* E&,4('*I"&%,(*!?LH*"8(*+(R*6&D&B(7(D*E&,4('*&,,/R*D('/,E.+9*78(*.+5/+'.'7(+5.('*0/4+)*0/D*!f\^H]A*WMP5,@*!f\^H]*5(,,'*0/D*XD/6(D7-*@A* &+)*0/D*hc32X*[g"??^*5(,,'*0/D*XD/6(D7-*SH*"]_Y*5(,,'*'7.,,*)/*+/7*'&7.'0-*XD/6(D7-*SH* *

F+7G74)#5?#*<;8#3L33#(8K,>75+).#M8<#+598#5?#65N4)*+8,2#;+5*874)#,46#5?#,#;5)7*7>8#?886(,-M#

955;#

37*.'*.B6/D7&+7*7/*+/7(*78&7*78(*'.9+.0.5&+7,-*).00(D(+7*68(+/7-6.5*D('6/+'('*/0*78(*).00(D(+7*5(,,*,.+('*&D(*.+*78(* B/)(,* '/,(,-* (N6,&.+()* %-* 7&Z.+9* .+7/* &55/4+7* /%'(DE()* ).00(D(+5('* .+* 78(* .+.7.&,* 5/+5(+7D&7./+'* /0* &* )/V(+* /0* Z(-*6D/7(.+'H*</*6&D&B(7(D*+(()'*7/*%(*58&+9()H*"8(D(0/D(*/+(*5&+*4'(*M21U?H]*R.78*+(R*6&D&B(7(D*E&,4('* I"&%,(*!?L*&+)*!"#*).&9D&B'*7/*.+E('7.9&7(*78(*.$!3!"%*/0*78(*).00(D(+7*%(8&E./D'*'8/R+*%-*5(,,*,.+('H*2'*+/7()*%-* 2,)D.)9(*(7*&,H*I@a??LA*/+(*.B6/D7&+7*Q4('7./+*.'*7/*).'7.+94.'8*R8(78(D*78(*).00(D(+7*%(8&E./D'*5&+*%(*(N6,&.+()* (N5,4'.E(,-* %-* ).00(D(+5('* .+* 46'7D(&B* 6D/7(.+* 5/+5(+7D&7./+'* /D* (N5,4'.E(,-* %-* ).00(D(+5('* .+* )/R+'7D(&B* 6D/7(.+*5/+5(+7D&7./+'A*/D*R8(78(D*&*5/B%.+&7./+*/0*46'7D(&B*&+)*)/R+'7D(&B*58&+9('*.'*+(()()H*3+)(()A*.7* 8&'*%((+*6D/6/'()*78&7*78(*B&.+*).00(D(+5('*%(7R((+*7-6(*3*&+)*33*%(8&E./D'*&D(*(''(+7.&,,-*)4(*7/*).00(D(+5('* .+* (00.5.(+5-* /0* 78(* .+.7.&7/D* 5&'6&'(* &57.E&7./+* %-* 78(* Y3!g* F]A>A;@GH* 37* 8&'* &,'/* %((+* 6D/6/'()* 78&7* 78(* B&.+* )(7(DB.+&+7*.'*78(*c32X*5/+5(+7D&7./+*D(,&7.E(*7/*5&'6&'($S*FSA;GH*2'*&66&D(+7*.+*78(*XD/6(D7-*@*).&9D&B*IO.94D(* ;LA*&+)*&'*&,'/*B(+7./+()*.+*2,)D.)9(*(7*&,H*I@a??L*%&'()*/+*Y#M*).&9D&B'A*.7*.'*5,(&D*78&7*).00(D(+5('*.+*c32X*/D* 5&'6&'($S* ,(E(,'A* 78&7* .'A* .+* )/R+'7D(&B* 6D/7(.+* 5/+5(+7D&7./+'A* 5&+* (N6,&.+* 78(* /%'(DE()* ).00(D(+5('* 0/D* [g"??^* &+)* !f\^H]* 5(,,* ,.+('H* "/* .+E('7.9&7(* R8(78(D* O#3X* &+)* 5&'6&'($>* ,(E(,'A* 78(* '/,(* 46'7D(&B* 6D/7(.+'* R8.58* .+.7.&,* 5/+5(+7D&7./+'* 58&+9(* 0/D* ).00(D(+7* 5(,,* ,.+('* .+* M21U?H]A* B&-* (N6,&.+* 78(* /%'(DE()* ).00(D(+5('* %(7R((+* 5(,,* 7-6('A* R(* 5/B647()* 78(* !"#* ).&9D&B'* '8/R.+9* 78(* (00(57'* /0* 58&+9('* .+* O#3X* /D* 5&'6&'($>* ,(E(,'* IO.94D(* >2LH* 2'* &66&D(+7* /+* 78(* ).&9D&BA* 58&+9.+9* O#3X* &+)* 5&'6&'($>* ,(E(,'* .+* [g"??^* 5(,,* ,.+(* 7/* B&758* 78(*

H HB S SB T TB

−0.5 0 0.5

Distribution of in silico predicted property 1 values

H HB S SB T TB

0 50

100 Percentage of property 1 satisfaction in cell population

H HB S SB T TB

False

True Expected vs in silico predicted property 1 satisfaction

H HX HB HBX S SB

−0.02 −0.01 0 0.01

Distribution of in silico predicted property 2 values

H HX HB HBX S SB

0 50

100 Percentage of property 2 satisfaction in cell population

H HX HB HBX S SB

False

True Expected vs in silico predicted property 2 satisfaction

H HX HBX T TB

−0.5 0 0.5

Distribution of in silico predicted property 3 values

H HX HBX T TB

0 50

100 Percentage of property 3 satisfaction in cell population

H HX HBX T TB

False

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