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Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
h ps://doi.o g/10.1186/s12964-025-02122-1 Cell Communica ion
and Signaling
†Pila Lice as-Boillos and Rósula Ga cia-Na as con ibu ed equally o
his wo k.
*Co espondence:
Eugenio San os
[email p o ec ed]
Fe nando C. Bal anás
cal [email protected]
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Abs ac
Backg ound Sos1 and Sos2 a e guanine-nucleo ide exchange ac o s o Ras and Rac small GTPases, which a e
in ol ed in a wide ange o cellula esponses including p oli e a ion and mig a ion. We ha e p e iously shown ha
Sos1 and Sos2 ha e di e en e ec s on cell mig a ion, bu he unde lying mechanisms a e no clea .
Me hods Using a 4-hyd oxy amoxi en-inducible condi ional Sos1KO mu a ion, he e we e alua ed he unc ional
speci ici y o edundancy o Sos1 and Sos2 ega ding he con ol o cell mig a ion and dynamics o ocal adhesions
(FAs) in p ima y mouse emb yonic ib oblas s (MEFs).
Resul s Func ional analysis o he ansc ip ome o p ima y Sos1/2WT, Sos1KO, Sos2KO and Sos1/2DKO-MEFs e ealed
a speci ic, dominan ole o Sos1 o e Sos2 in ansc ip ional egula ion. Sos1KO MEFs had an inc eased numbe and
s abili y o ocal adhesions (FAs) and cu bed p o usion and sp eading. Con e sely, Sos2KO MEFs displayed uns able
FAs wi h inc eased p o usion. In e es ingly, Sos1, bu no Sos2, abla ion educed he le els o GTP-bound Rac a he
leading edge. In 3D, howe e , only Sos1/2KO MEFs showed inc eased in asion and ma ix deg ada i e capaci y, which
co ela ed wi h inc eased exp ession o he Mmp2 and Mmp9 gela inases. Mo eo e , inc eased ma ix deg ada ion in
Sos1/2KO MEFs was ab oga ed by ea men wi h Mmp2/9 inhibi o s.
Conclusions Ou da a demons a e ha Sos1 and Sos2 ha e di e en unc ions in FAs dis ibu ion and dynamics in
2D whe eas in 3D hey ac oge he o egula e in asion and un eil a p e iously undesc ibed mechanis ic connec ion
be ween Sos1/2 and he egula ion o Mmp2/9 exp ession in p ima y MEFs.
Sos1 abla ion al e s ocal adhesion dynamics
and inc eases Mmp2/9-dependen gela inase
ac i i y in p ima y mouse emb yonic
ib oblas s
Pila Lice as-Boillos1†, RósulaGa cia-Na as1†, Cla aLlo en e-González2, L. F anciscoLo enzo-Ma in3, LuisLuna-
Ramí ez4, RocíoFuen es-Ma eos1, Nu iaCalzada1, F ancisco M.Vega5, Ma k R.Hol 6, Anne J.Ridley7, Xose R.Bus elo8,
MiguelVicen e-Manzana es2, EugenioSan os1* and Fe nando C.Bal anás1,4*
Page 2 o 18Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
In oduc ion
Ras and Rac p o eins a e small GTPases ha con ol a
wide a ie y o cellula p ocesses, including cellula p o-
li e a ion, di e en ia ion, mig a ion and su i al. These
p o eins swi ch be ween inac i e (GDP-bound) and
ac i e (GTP-bound) con o ma ions. GTP binding and
i s hyd olysis a e modula ed by nega i e and posi i e
egula o s. GAPs (GTPase-ac i a ing p o eins) igge
GTP hyd olysis by Ras/Rac, hus inac i a ing hem. Con-
e sely, GEFs (Guanine nucleo ide exchange ac o s) ca -
alyze GTP binding, ac i a ing hem. Sos (Son o se enless)
p o eins, Sos1 and Sos2, a e he mos widely exp essed
and unc ionally ele an Ras GEFs [1–5]. In addi ion o
Ras, Sos1/2 ac i a e o he ela ed GTPases, including Rac
[4–8].
Despi e hei s uc u al homology, he unc ional p op-
e ies o Sos1 and Sos2 a e ma kedly di e en . P io anal-
yses o cons i u i e knockou (KO) s ains demons a ed
ha Sos1−/− mice die du ing mid-emb yonic ges a ion
[6]. Gene a ion o loxed Sos1/2 mice [9, 10] allowed
e alua ion o he oles o Sos1/2 in di e en cell lines,
issues and o gans unde physiological and pa hologi-
cal condi ions [8, 11–22]. Mos o he s udies ha e been
ocused on iden i ying he essen ial ole o Sos p o eins,
and in pa icula Sos1, in cellula p oli e a ion [4]. P i-
ma y mouse emb yonic ib oblas s (MEFs) om Sos1KO,
Sos2KO and Sos1/2DKO e ealed he p e alen ole o Sos1
o e Sos2 in oxida i e, s ess-dependen egula ion o cell
p oli e a ion [12, 18]. These s udies laid he ounda ion
o he iden i ica ion o Sos1 as a po en ial he apeu ic
a ge o ea cance [4, 11, 14–16, 22–25].
Independen o hei ole in cell p oli e a ion, Sos1/2
deple ion in MEFs impai s cell mig a ion in 2D wound
healing assays [18, 26]. Simila obse a ions we e made
in o he lineages such as lymphocy es, mac ophages o
muscle cells [27–29]. Howe e , mo e de ailed s udies a e
needed o p ecisely de ine he speci ic unc ional con i-
bu ions o Sos1/2 p o eins in he egula ion o cell mig a-
ion o adhesion.
He e, we use Sos1KO, Sos2KO and Sos1/2DKO p ima y
MEFs o demons a e ha Sos1 is a c i ical media o o
he dis ibu ion and dynamics o ocal adhesions (FAs)
as well as he ec ui men o GTP-loaded, ac i a ed Rac
o he p o uding edge o mig a ing cells. Unexpec edly,
Sos1/2 abla ion in p ima y MEFs inc eased hei abil-
i y o in ade in 3D and deg ade gela in, which is depen-
den on he inc eased exp ession o Mmp2 and 9. Sos1/2
deple ion in p ima y MEFs inc eased bo h Mmp2/9 gene
and p o ein exp ession as well as he Mmp2/9-dependen
capaci y o deg ade gela in.
Toge he , hese da a indica e ha Sos1, and Sos2 o a
lesse ex en , con ol FA dis ibu ion and dynamics and
e eal hei c ucial ole in 3D mig a ion h ough con ol-
ling he exp ession o ma ix me allop o einases.
Ma e ials and me hods
Animal models
A mouse s ain ha bo ing a loxed e sion o Sos1 gene
wi h exon 10 lanked by LoxP si es (Sos1 l/ l) [9] was
c ossed wi h mice exp essing a TAM-inducible C e
ecombinase downs eam o he RERT (Jackson Labo-
a o ies; s ock numbe 017585, exp essing an inducible
C e-ERT2, C e ecombinase used o a iple mu an
o m o he human es ogen ecep o , om he endog-
enous Pol 2a locus) p omo e o gene a e homozygous.
Sos1 l. − C e/Sos1 l. − C e mice, which we e hen ma ed wi h
cons i u i e Sos2KO mice [30]. Resul ing he e ozygous
mice we e subsequen ly in e b ed o gene a e ou dis-
inc geno ypes used in his epo : con ol (Sos1/2WT),
Sos1 single-KO (Sos1KO), Sos2 single-KO (Sos2KO), and
Sos1/2DKO [10]. Animals we e main ained and eu ha-
nized in he NUCLEUS animal acili y o he Uni e si y
o Salamanca acco ding o Eu opean (2007/526/CE)
and Spanish (RD 1201/2005 and Law 32/2007) laws. All
expe imen s we e app o ed by he Bioe hics Commi ee
o he Cance Resea ch Cen e (#417).
P ima y MEF isola ion and cul u e
Sos1WT, Sos1KO, Sos2KO and Sos1/2DKO p ima y MEFs
we e isola ed om E13.5 emb yos o he co espond-
ing geno ypes and used as p ima y (low passage) cul-
u ed cells as p e iously desc ibed [12]. All expe imen al
g oups we e ea ed wi h 4OHT a he indica ed ime
poin s a each case (H6278, 0.3 µM, Me ck Sigma-
Ald ich, USA) in DMEM wi h 10% FBS, glu amine and
an ibio ics, unde iden ical condi ions o induce Sos1
abla ion in Sos1KO and Sos1/2DKO expe imen al g oups
and o exclude any possible o - a ge e ec s in Sos1/2WT
and Sos2KO expe imen al g oups. Cells we e ou inely
es ed o Mycoplasma (# ep-p 1, PlasmoTes Myco-
plasma De ec ion Ki ; In i oGen).
Incucy e
A o al o 1,500 cells pe well o DMSO o BI-
3406- ea ed (1 µM) o 24h we e seeded in 96-well cul-
u e pla es and allowed o g ow o e nigh . Cell g ow h
and mo phology we e moni o ed by using he Incucy e®
SX5 Li e-Cell Analysis Sys em and analyzed wi h he AI
Cell Heal h Analysis So wa e Module, which enabled
cell iden i ica ion and a ea measu emen . Readings we e
aken e e y 6h a 20× magni ica ion.
Keywo ds Sos1, Sos2, RasGEF, Ras, Rac, Mig a ion, Mmp2/9
Page 3 o 18Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
Mic oa ay analysis and RT-qPCR assay
MEFs we e ea ed wi h 4OHT o 12 days hen RNA was
isola ed om subcon luen MEFs wi h Nyzol and QIA-
GEN RNeasy Mini ki (74104, QIAGEN) ollowing he
manu ac u e ’s indica ions. Pu i ied RNA was hyb id-
ized o A yme ix “Cla iom S” mouse a ays ollowing
he manu ac u e ’s p o ocol. All da a ha e been uploaded
and a e accessible a he NCBI Gene Exp ession Omni-
bus (GEO) da abase (GSE277505). R e sion 3.6.3 was
used o s a is ical analyses along wi h Py hon e sion
3.9 o ex ile p ocessing. Values o signal in ensi y we e
ob ained om exp ession mic oa ay CEL iles a e
obus mul ichip a e age (RMA). Di e en ially exp essed
genes we e iden i ied using linea models o mic oa ay
da a (Limma). Adjus ed P- alues o mul iple compa i-
sons we e calcula ed applying he Benjamini-Hochbe g
co ec ion (FDR). Gene On ology pa hway en ichmen
analyses we e pe o med using DAVID. Exp ession hea -
maps we e c ea ed using he hea map3 R package.
mRNA exp ession le els we e de e mined by quan-
i a i e RT-PCR analysis o RNA samples ex ac ed
om MEFs. 1 µg o RNA was e e se- ansc ibed o
cDNA (High Capaci y cDNA Re e se T ansc ip ion
Ki ; #4368814, Li e Technologies). Final concen a-
ions o cDNA we e measu ed (Nanod op Technologies
ND-1000) and adjus ed o 0.5µg/µl. Fo qRT-PCR analy-
ses, he ollowing gene-speci ic SYBRG een-based p im-
e s we e used: Mmp2 Fw 5′-caagga ggac cc ggcaca -3′
and Mmp2 R 5′- ac cgcca cagcg ccca -3′, Mmp9 Fw
5′-gc gac acga aaggacggca-3′ and Mmp9 R 5′- ag gg g-
caggcagag agga-3′, β-ac in Fw 5′-cagcc cc c ggg a g-3´
and R 5′-cagcc cc c ggg a g-3´.
The exp ession le els o β-ac in we e used as an in e -
nal con ol o no maliza ion. The da a we e g aphically
displayed using G aphPad P ism 8. S a is ical signi icance
was es ablished a p < 0.05.
Immuno luo escence
MEFs we e ea ed o 12 days wi h 4OHT hen ixed
wi h pa a o maldehyde (4%) o 10min and incuba ed
wi h mouse an i-Paxillin an ibody (1:1000, 05-417, clone
5H11; Me ck Millipo e, Bille ica, USA) o e nigh a 4ºC.
Cells we e hen incuba ed wi h Texas Red-conjuga ed
an i-mouse an ibody (1:500, Jackson Immuno-Resea ch,
Wes G o e, PA, USA) o 45min and coun e s ained
wi h 4,6-diamidino-2-phenylindole (1:1000; DAPI) and
Alexa Fluo 488-conjuga ed phalloidin (1:1000, A12379,
The mo Fishe Scien i ic (In i ogen), UK) a oom em-
pe a u e. Images we e acqui ed wi h a con ocal lase -
scanning mic oscope (Leica SP5, We zla , Ge many).
To speci ically de ec Rac ac i i y by immuno luo es-
cence, 4OHT- ea ed (12d) MEFs we e g own on 22mm
ype I collagen-coa ed co e slips (354089 BD Biosci-
ences) o 100% con luency. A e 24h, in i o sc a ch
assays we e pe o med as desc ibed p e iously [18] wi h
a p10 pipe e ip. Cells we e ixed 30min a e he sc a ch
in 4% PFA o 15min a 37ºC. Fixed cells we e washed
wice wi h PBS, pe meabilized wi h 0.1% T i on X-100
(Me ck Sigma-Ald ich) in PBS o 10 min and incu-
ba ed wi h blocking solu ion con aining 5% BSA, 2% goa
se um, 0.1% T i on X-100 (in PBS) o 1h a RT. Co -
e slips we e incuba ed wi h 0.3mg/ml GST-CRIB pu i-
ied p o ein (Addgene, Ca #12217) o 1h a RT, washed
h ee imes wi h PBS and incuba ed wi h mouse an i-
GST an ibody (1:100, sc-138, San a C uz Bio echnology)
o e nigh a 4ºC in an ibody solu ion (2% BSA, 2% goa
se um and 0.1% T i on in PBS). Co e slips we e hen
washed h ee imes wi h PBS and incuba ed wi h Cy3
an i-mouse an ibody (1:500, Jackson ImmunoResea ch)
and coun e s ained wi h DAPI (1:1000) o 1h a RT in
an ibody solu ion. A e s aining, co e slips we e washed
h ee imes in PBS and moun ed wi h an i- ading eagen
(P36970, Li e Technologies). Images we e acqui ed using
a Leica DM6000B mic oscope.
In e e ence e lec ion mic oscopy
In e e ence e lec ion mic oscopy (IRM) uses pola ized
ligh o o m an image o cells on a glass su ace and he
in ensi y o he signal p o ides a measu emen o e en s
happening a he in e ace o he cell memb ane wi h he
subs a um, including ocal adhesions, which appea as
da k pa ches due o close apposi ion o he subs a um,
and o he si es o ocal con ac s including in lamellipo-
dia [31, 32]. MEFs we e ea ed wi h 4OHT o 9 days
and hen 1.5 × 105 cells pe geno ype we e seeded on
ib onec in-coa ed (10µg/ml) 2-cm diame e co e slips.
24h a e pla ing he cells, using a Zeiss LSM510 con o-
cal mic oscope, ime-lapse sequences o images (e e y
20s o 1h) we e aken o gene a e adhesion maps. The
images we e p ocessed so ha pixels ep esen ing FAs
s abili y we e colo ed, ed, g een o blue co espond-
ing o ocal adhesions p esen ea ly du ing imaging ( ed)
h ough o ocal adhesions p esen la e du ing imaging
(blue), as p e iously desc ibed [31]. Whe e con ac s we e
s able, he o e lapping ames esul ed in a p eponde -
ance o whi e o ligh g ey as a esul o me ging o he
base colo . Using ZEN so wa e (Zeiss) he numbe o
lamellipodia and FAs, he a ea o ex ension and he s a-
bili y o FAs we e measu ed and quan i ied using ImageJ
so wa e (NIH 2.0.0).
Using his e y same me hodology, we we e also able o
e alua e cell sp eading o indi idual MEFs om he ou
di e en geno ypes. To do ha , we o e lapped all images
aken o each indi idual cells (one image e e y 20s o
1h), hen gene a ing an image ha allowed o examine
and measu e he sp eading o he cell h ough he ime.
Page 4 o 18Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
Gela in deg ada ion assay
Gela in deg ada ion assays we e pe o med as p e iously
desc ibed [33]. B ie ly, MEFs we e ea ed wi h 4OHT o
12 days o wi h he Sos1 inhibi o BI-3406 o 24h (1µM,
HY-125817, MedChemExp ess), hen 3 × 105 cells pe
geno ype we e seeded on co e slips coa ed wi h FITC-
conjuga ed gela in (1 mg/ml; M1303, Bio ision, CA,
USA) as p e iously desc ibed [33]. A e 8h, cells we e
ixed in 4% PFA o 15min and subsequen ly washed
wi h PBS and incuba ed in BSA solu ion (3% in PBS con-
aining 0.1% T i on X-100) o 30min in he da k a oom
empe a u e (RT). The BSA solu ion was hen emo ed,
and samples we e s ained wi h Alexa Fluo 568 phalloi-
din (1:500 in PBS con aining 0.3% BSA and 0.1% T i on
X-100) o 1h a RT, p o ec ed om ligh . A e wash-
ing wi h PBS, co e slips we e moun ed wi h an i ading
solu ion medium con aining DAPI (1:1000). Images we e
aken wi h a lase -scanning con ocal mic oscope (Leica
SP5, We zla , Ge many). To quan i y gela in deg ada-
ion, he a ea ac ion (% a ea ha co esponds o deg-
ada ion) was measu ed in black and whi e images using
ImageJ (NIH 2.0.0). Values we e no malized o he
numbe o DAPI-s ained nuclei.
The e ec o he gela inase inhibi o s Ilomas a
(S715702, Selleckchem) and ARP-100 (704888-90-4,
San a C uz Bio echnology, USA) on gela in deg ada ion
was e alua ed by pla ing MEFs on FITC-conjuga ed gel-
a in-coa ed dishes as abo e. Th ee hou s a e pla ing ( o
allow he a achmen o he cells), Ilomas a (0.5 nM) o
ARP-100 (7.5 µM) we e added o he medium in se um-
ee DMEM. A e 8h, MEFs we e ixed in 4% PFA and
analysed o gela in deg ada ion as desc ibed abo e.
Wound healing assay
In i o sc a ch wound assay was pe o med as p e i-
ously desc ibed [18] using FIJI so wa e (NIH 2.0.0) o
ace mo emen o indi idual cells. B ie ly, WT p ima y
MEFs we e ea ed wi h ehicle (DMSO), BI-3406 (1µM),
o he Rac inhibi o 1A-116 (10µM; HY-104064, Med-
ChemExp ess) o 24h, and hen he con luen cells o
bo h condi ions we e sc a ched wi h a mic opipe e ip
and closu e o he wounded a ea was eco ded o 24h by
using in e ed phase-con as pho omic oscopy (Nikon
Eclipse Ti-E).
In e ed in asion assay
In e ed in asion assays we e pe o med as p e iously
desc ibed [34, 35]. B ie ly, 4–5mg/ml Ma igel (BD Bio-
sciences, #354234) was dilu ed 1:1 in cold PBS. Then,
100µl o dilu ed Ma igel was allowed o polyme ize in
8-µm po e answell inse s (Co ning, #353097) o 1h
a 37ºC. Inse s we e hen in e ed, and a o al o 6 × 104
p ima y MEFs (p e- ea ed wi h 4OHT o 9 days) o
each geno ype we e seeded on o he ou e il e su ace
and allowed o adhe e o 4h. To emo e una ached cells
and FBS-con aining medium, inse s we e washed by
dipping in se um- ee DMEM and hen placed in 500µl
se um- ee DMEM con aining 0.3 µM o 4OHT as p e-
iously desc ibed [12]. 100µl o 10% FBS-supplemen ed
medium we e added on op o he solidi ied Ma igel/PBS
mix u e o c ea e he chemo ac ic g adien . Addi ion-
ally, ano he se o cells we e ea ed wi h Ilomas a (0.5
nM) o ARP-100 (7.5 µM). A e 72h, cells we e s ained
wi h 4 µM Calcein-AM iabili y ma ke (C3100MP, In i-
ogen) o 1h a 37ºC. Cells ha did no c oss he il e
we e emo ed wi h a co on swabs.
In ading cells we e imaged in a Leica SP5 con ocal
mic oscope using a 20× objec i e. Se ial op ical sec ions
we e cap u ed a 2.5u m in e als. The a ea co e ed by
cells was measu ed in each sec ion using he Fiji plugin
“A eaCalcula o ” in 8-bi images ( h eshold 20/255). Rel-
a i e in asion was calcula ed as he a ea co e ed by cells
a each dep h o he Z-s ack. Two independen expe i-
men s in duplica e we e pe o med o each sample.
Wes e n blo ing
Subcon luen MEFs ha had been ea ed wi h 4OHT
o 12 days o wi h BI-3406 (1 µM) o 24h, we e lysed
wi h RIPA bu e . In all cases, 30 µg o o al p o ein
we e loaded in elec opho esis gels, and immunoblo -
ing was pe o med as p e iously desc ibed [21]. P i-
ma y an ibodies used we e: abbi an i-Mmp2 (#4060,
1:1000, Cell Signaling), mouse an i-Sos1 (610096, 1:500,
BD, USA), abbi an i-Sos2 (sc-15358, 1:500, San a C uz
Bio echnology) mouse an i-phospho-Myosin ligh chain
2 (#3675, 1:1000, Cell Signaling), abbi an i-Myosin
(#8505, 1:2000, Cell Signaling), abbi an i-phospho Fak
(#sc11765-R, 1:100, San a C uz Bio echnology), mouse
an i-Fak (#62220, 1:1000, Cell Signaling) and mouse an i-
Tubulin (T5293, 1:10000, Sigma). Seconda y an i-mouse
o an i- abbi an ibodies include goa an i-mouse sec-
onda y an ibodies (In i ogen, Ca #A21057; #35521) and
goa an i- abbi seconda y an ibodies (GE Heal hca e,
Ca #A21076; #35571).
S a is ical analysis
G aphPad P ism ( 8.0.1, G aphPad Inc, USA) so wa e
was used. One-way ANOVA and Bon e oni’s es s we e
applied o pa ame ical da a, and K uskal–Wallis and
Mann-Whi ney U- es o non-pa ame ical da a. Fo
compa isons be ween ehicle and BI-3406- ea ed cells,
S uden ’s es was used. Signi ican di e ences we e
conside ed a p alue < 0.05.
Page 5 o 18Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
Resul s
Signi ican ansc ip ional impac o he abla ion o Sos1,
bu no Sos2, on he ansc ip ional signa u e o p ima y
mouse emb yo ib oblas s
Ou p e ious s udies showed ha , in p ima y MEFs,
Sos1/2 a e c i ical media o s o cell p oli e a ion and
mig a ion [12, 18]. To in es iga e how Sos1 and Sos2
migh con ibu e o mig a ion, we ini ially examined he
e ec o Sos1 and Sos2 gene ic abla ion (indi idually o
combined) on he ansc ip ional signa u e o p ima y
MEFs using mic oa ay hyb idiza ion assays (Fig. 1).
Mul iclass compa isons among he lis s o di e en ially
exp essed genes ob ained unde s anda d cu o alues
(FDR = 0.05) e ealed d ama ic di e ences in he an-
sc ip omic p o ile o con ol Sos1/2WT MEFs om Sos-
deple ed (Sos1KO, Sos2KO and Sos1/2DKO) MEFs (Fig.1A).
Abla ion o Sos2 alone had he leas signi ican e ec on
he ansc ip ional signa u e o p ima y MEFs, wi h only
59 di e en ially exp essed genes ela i e o hei con ol
Sos1/2WT coun e pa s (Fig. 1A). In con as , genomic
dis up ion o Sos1, alone o in combina ion wi h Sos2,
ma kedly al e ed he ansc ip ional p o ile o p ima y
MEFs (Fig.1A), wi h 2305 di e en ially exp essed genes
when Sos1 was indi idually abla ed and a o al o 2506
genes we e di e en ially exp essed when bo h Sos iso-
o ms we e dele ed (Fig. 1A). Analysis wi h Venn dia-
g ams e ealed ha he p o ile o di e en ially exp essed
genes in Sos1/2DKO MEFS in ol ed mo e signi ican o e -
lapping wi h ha o Sos1KO han wi h Sos2KO p ima y
MEFs (Fig.1B). These esul s a e consis en wi h a mo e
impo an ole o Sos1 o e Sos2 in al e ing ansc ip ion
in p ima y MEFs. Howe e , he addi i e e ec obse ed
in Sos1/2DKO cells compa ed o Sos1KO cells sugges ed
he possibili y o a po en ial, pa ial unc ional edun-
dancy o Sos2 ha only becomes de ec able when Sos1
is absen .
Fig. 1 Sos1, bu no Sos2, deple ion signi ican ly a ec s he ansc ip ional signa u e o p ima y MEFs. (A) Pai wise compa isons o di e en ial gene ex-
p ession among di e en Sos1/2 geno ypes. A se o 12 independen chip mic oa ay hyb idiza ions (n = 3 independen sample pe geno ype) we e pe -
o med using RNA ex ac ed om ac i ely g owing p ima y MEFs belonging o he indica ed geno ypes. The hea maps show he esul s o hie a chical
clus e ing and mul iclass compa isons iden i ying he up egula ed ( ed) and down egula ed (blue) p obese s ha showed signi ican di e en ial exp es-
sion a s ingen condi ions (FDR = 0.05) when compa ing Sos1/2WT p ima y MEFs wi h he es o expe imen al g oups. To al numbe o ep essed (blue)
and up egula ed ( ed) di e en ially exp essed p obese s de ec ed in hese compa isons is indica ed on op o each hea map. Values in each ho izon al
cha indica e -Log P. (B) Venn diag ams display he ela ions be ween he lis s o di e en ially exp essed genes (DEGs) in he h ee se s o compa isons.
(C) Func ional anno a ion o DEGs. Tex on he le side o he g aphs iden i y unc ional ca ego ies ha a e en iched a ele a ed s a is ical signi icance
wi hin he lis s o DEGs ( ed: o e exp essed; blue: ep essed) included in he clus e s iden i ied in Sos1/2WT-Sos1KO and Sos1/2WT-Sos1/2DKO pai wise
compa isons. The ba s ep esen he pe cen age o he o al numbe o di e en ially exp essed ep essed (blue) o o e exp essed ( ed) gene p obe se s
asc ibed o speci ic g oups o genes o each o he abo e dend og ams ha we e iden i ied as signi ican ly en iched (hype geome ic p- alues in i alics)
Page 6 o 18Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
Func ional anno a ion analysis o he di e en ially
exp essed genes in he dend og ams showed ha he
subse o ep essed genes in Sos1KO p ima y MEFs,
which include bo h Sos1KO and Sos1/2DKO cells, is sig-
ni ican ly en iched in unc ional GO ca ego ies gene -
ally linked wi h cell cycle and cell di ision oge he wi h
mic o ubule cy oskele on o ganiza ion (Fig. 1C). On
he o he hand, clus e s con aining up egula ed genes
in Sos1KO MEFs included genes linked o ex acellula
ma ix o ganiza ion and egula ion o cell adhesion, as
well as nega i e egula ion o cell mig a ion (Fig.1C). In
con as , di e en ially exp essed genes in Sos2KO MEFs
did no display unc ional clus e ing.
O e all, ou da a e eal ha Sos1 abla ion induces
much mo e signi ican al e a ions o he MEF ansc ip-
ional p o ile han Sos2, and ha a majo subse o genes
al e ed by he absence o Sos1 include egula o s o he
cellula cy oskele on and cell mig a ion.
Sos1/2 deple ion a ec s cellula mo phology and ocal
adhesion dis ibu ion and dynamics
We ha e epo ed p e iously ha Sos1 lack o exp es-
sion, indi idually o combined wi h Sos2, al e ed he
ac in cy oskele on and impai ed MEF p oli e a ion [12,
18]. Consis en ly, ansc ip omics analysis o Sos1KO
MEFs e ealed an up egula ion o genes in ol ed in he
ep ession o cell mig a ion, co ela ing wi h a signi ican
educ ion o he abili y o hese cells o mig a e in in i o
and in i o wound-healing assays [18, 19].
Cell mig a ion is coo dina ed by dynamic changes
o he ac in cy oskele on and in eg in-media ed FAs
[36, 37]. To unde s and how Sos1/Sos2 p o ein deple-
ion educes cell mig a ion, we analyzed he e ec s o
Sos1 and Sos2 abla ion on he dis ibu ion and dynam-
ics o FAs and he ac in cy oskele on (Fig. 2). Sos1KO
and Sos1/2DKO MEFs displayed an abe an dis ibu-
ion o ac in cables, which we e sho e and mo e adial,
wi h equen e ac ion edges ha esul ed in appa -
en ly smalle , isome ic, and la ened cells. These ac in
ibe s we e capped by sho FAs h oughou he pe iph-
e y o he cell as well as mo e cen al posi ions, e ealed
by s aining wi h an an i-paxillin an ibody. By con as ,
Sos1/2WT and Sos2KO MEFs displayed a egula elon-
ga ed ib oblas ic mo phology including an abundance o
shape-de ining s ess ibe s ending in long FAs (Fig.2). In
hese cells, FAs we e mainly es ic ed o he wo main
poles o he cells (Fig.2, whi e a ows).
We used in e e ence e lec ion mic oscopy (IRM) o
analyse dynamic changes in si es o closes con ac wi h
he subs a um, which ep esen FAs as well as smalle
adhesi e si es in lamellipodia [31]. FAs in bo h WT and
Sos2KO MEFs we e mainly es ic ed o he poles o elon-
ga ed cells, showing less s ong signal in compa ison
wi h Sos1-de oid MEFs and displaying mo phologies
mainly pa allel o he longi udinal axis (Fig.3A, B). In
con as , FAs in Sos1KO MEFs (Sos1KO and Sos1/2DKO)
we e obse ed a ound he whole ci cula cell pe ime e
and wi h a andom o ganiza ion (Fig.3A, B). Quan i a-
i e measu emen s e ealed ha he numbe o FAs
pe cell was signi ican ly highe in Sos1KO MEFs han
in Sos1/2WT and Sos2KO MEFs (Fig. 3F). In e es ingly,
Sos2KO MEFs displayed a modes educ ion in he num-
be o FAs compa ed o con ol MEFs (Fig.3F). These
da a sugges ed ha Sos1 dele ion impai ed adhesion
ma u a ion. To add ess his, we gene a ed pseudo-col-
o ed IRM images ep esen ing he posi ion o he cell
con ac s a he beginning o he analysis (in ed), a hal
ime (in g een) and a he end (in blue) o he eco ding.
Whi e colo indica es a supe posi ion o ed/g een/blue
colo s and ep esen s a s a ic FA (Fig.3E). We obse ed
ha bo h Sos1/2WT and Sos2KO MEFs displayed mo e
dynamic cell con ac s consis en wi h a pola ized mig a-
o y beha io , whe eas Sos1KO MEFs exhibi ed a mo e
s a ic condi ion, wi h non-pola ized mo ili y o he cell
memb ane (Fig.3E). Mo eo e , Sos1/2DKO MEFs ba ely
modi ied hei con ac posi ions (Fig.3E, no e he mos ly
whi e display), indica ing ha concomi an Sos1/2 deple-
ion se e ely a ec ed he dynamics o FAs and he mig a-
o y abili y in MEFs. We nex hypo hesized ha impai ed
adhesion ma u a ion would impai cell sp eading. Cell
a ea analysis e ealed ha Sos1KO cells we e sligh ly
smalle han con ol and Sos2KO cells (Fig.3H), indica -
ing a de icien abili y o sp ead. Consis en ly, dynamic
analysis o he numbe o ex ended lamellipodia, indica-
i e o p o usi e mig a ion, pe cell e ealed ha Sos1KO
cells displayed a signi ican ly lowe numbe o p o u-
sions han con ol o Sos2KO cells, which in u n had a
sligh ly highe numbe o p o usions han con ol cells
(Fig.3I).
Taken oge he , ou obse a ions indica e ha Sos1
deple ion in MEFs, alone o in combina ion wi h Sos2
dele ion, esul s in an inc ease in he numbe o FAs
and ewe lamellipodia pe cell ha co ela e wi h a
high p opo ion o s able FAs and a educ ion in he cell
sp ead a ea, impai ing cell sp eading and hence he abil-
i y o he cell o mig a e. Ou da a also indica e a speci ic
ole o Sos2 in memb ane p o usion and cell adhe-
sion ha is supe seded when bo h iso o ms a e dele ed
simul aneously.
Sos1 abla ion speci ically al e s he localiza ion o
ac i a ed Rac in F-ac in ich domains and dec eases
cellula con ac ili y
In addi ion o Ras, Sos1 ac i a es Rac [7, 8, 38]. While
he exac mechanism o Rac ac i a ion by Sos1 is s ill
poo ly unde s ood, some da a poin o he c ucial ole
o Sos1 in he con ol o lamellipodial p o usion, cell
mig a ion and in asion [4, 7]. Using SosKO MEFs we
Page 7 o 18Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
Fig. 2 Sos1 deple ion al e s ocal adhesion and F-ac in dis ibu ion in p ima y MEFs. Rep esen a i e con ocal mic oscopy images o indi idual p ima y
MEFs om he ou geno ypes (Sos1/2WT, Sos1KO, Sos2KO and Sos1/2DKO) s ained wi h o ac in ilamen s wi h phalloidin (g een), ocal adhesions (FA) wi h
an i-Paxillin an ibody ( ed) and o nuclei wi h DAPI (blue). A ows show p edominan a eas o FA localiza ion in Sos1/2WT and Sos2KO MEFs. Scale ba :
25μm
ha e p e iously shown ha he global le els o cellula
GTP-bound Rac (in pull-down assays) emained unal-
e ed upon indi idual o combined Sos1/2 deple ion
when compa ed wi h WT con ol cells [18]. Howe e ,
Sos1 could con ol he spa ial dynamics o Rac ac i-
a ion while ha ing no e ec on he global le els o
ac i a ed Rac. To add ess his possibili y, we localized
ac i e Rac-GTP by immuno luo escence in Sos1/2KO
MEFs upon wound healing (Fig. 4). These expe i-
men s e ealed ha Rac-GTP clus e s a he ee edges
o Sos1/2WT and Sos2KO MEFs (Fig. 4). Con e sely,
Sos1KO and Sos1/2DKO MEFs did no display Rac-GTP
clus e ing a ee edges, being only de ec able in he
cy oplasm o cells (Fig.4).
Since p o usion and e ac ion a e igh ly coo dina ed
o media e cell mig a ion [37], we also examined he con-
ac ile s a us o he cell using phospho yla ion o he
egula o y ligh chain o myosin II as a iducia y ma ke
o con ac ion [39]. We obse ed a ma ked educ ion
in he le els o phospho-myosin ligh chain 2 (Se 19)
in Sos1KO MEFs (Sos1KO and Sos1/2DKO) compa ed o
Sos1/2WT and Sos2KO MEFs (Fig.5). These da a co ela e
well wi h he obse ed mo phological e ec s o Sos1KO
MEFs (Figs.1 and 2) and wi h he epo ed e ec o he
educ ion o he le els o RLC o i s ac i a ion in mesen-
chymal cells [40]. O e all, hese esul s sugges ha Sos1
is equi ed o he local ac i a ion o Rac a p ospec i e
p o uding edges o mig a ing cells. Gi en ha , his e ec
is likely con ined o mig a ing cells, which comp ise
a small pe cen age o he o al cells in a wound healing
assay, hese esul s a e compa ible wi h he lack o e ec
o Sos1 deple ion in he global le els o Rac ac i a ion, as
epo ed p e iously [10].
Page 8 o 18Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
Fig. 3 (See legend on nex page.)
Page 9 o 18Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
Sos1/2 deple ion acili a es in asi e mig a ion o MEFs in
3D cul u e
In 2D, Sos1 is c ucial o cell mig a ion in a Rac-depen-
den manne [18]. In his ega d, ou esul s demon-
s a ed ha Rac inhibi ion esul ed in simila impai men
in wound closu e as de ec ed wi h he Sos1 inhibi o ,
BI-3406 (Supplemen a y Fig.1). Howe e , mul iple s ud-
ies ha e highligh ed he la ge di e ences in signaling,
adhesion, ac ion and ma ix eo ganiza ion in cells
mig a ing in 3D ( e iewed in [41]). To s udy he beha io
o SosKO MEFs in 3D mig a ion, we pe o med in e ed
Ma igel in asion assays [35]. Su p isingly, bo h Sos1
o Sos2 abla ion, indi idually o combined, signi ican ly
inc eased he in asion o a 3D Ma igel ma ix when
compa ed o hei Sos1/2WT coun e pa s (Fig. 6). In
addi ion, Sos2KO cells ended o in ade less han Sos1
lacking MEFs (Fig.6).
While 2D mig a ion depends almos exclusi ely on
cycles o memb ane p o usion and cell body e ac ion
[42], 3D mig a ion o mesenchymal cells also equi es
ma ix eo ganiza ion and deg ada ion [43]. The oppo-
si e e ec o Sos1 deple ion in 3D mig a ion compa ed
o i s e ec in 2D sugges ed ha i s ole in p o usion/
e ac ion could be supe seded by ano he ype o de ec .
To add ess his, we measu ed he le els o Mmp2/9
(Mmp2 and Mmp9 genes), which a e canonical ma ix
me allop o eases in ol ed in he deg ada ion o colla-
gens and some o he ma ix p o eins du ing 3D mig a-
ion, including collagen IV, which is p esen in Ma igel
[44–46]. These expe imen s e ealed ha Mmp2 mRNA
was signi ican ly o e exp essed in Sos1/2DKO cells when
compa ed o he es o expe imen al g oups (Fig.6A).
Con e sely, indi idual Sos1 o Sos2 deple ion had a
much lowe , bu s ill signi ican , e ec on Mmp2 exp es-
sion (Fig.6A). Wes e n blo expe imen s also e ealed
inc eased exp ession o Mmp2 p o ein (Fig. 6B). On
he o he hand, indi idual abla ion o Sos1 and Sos2
inc eased exp ession o Mmp9, bu he simul aneous
deple ion o Sos1 and Sos2 had almos no e ec com-
pa ed o WT MEFs (Fig.6A). These da a sugges ha
Sos1 and Sos2 no mally supp ess Mmp2 and Mmp9
ansc ip ion, and hus deple ion o ei he Sos1 o Sos2
inc eases Mmp2 and Mmp9 le els.
Sos1/2 abla ion inc eases gela in deg ada ion ac i i y in
MEFs
To es whe he Sos1/2 deple ion inc eased he abili y
o MEFs o deg ade ex acellula ma ix in a Mmp2/9-
dependen manne , we s udied he e ec o Sos1/2 deple-
ion in a 2D gela in deg ada ion assay (Fig.7). We ound
ha single o combined Sos1/2 deple ion signi ican ly
inc eased he abili y o MEFs o deg ade gela in as com-
pa ed o WT MEFs (Fig.7A). In e es ingly, Sos1KO MEFs
exhibi ed a sligh ly highe abili y o deg ade gela in han
hei Sos2KO coun e pa s (Fig.7A).
We es ed whe he gela in deg ada ion was depen-
den on Mmp2/9 by ea ing he cells wi h selec i e
inhibi o s o hese Mmps (ARP-100 and Ilomas a ,
Fig.7B and C, espec i ely). We ound ha hese Mmp
inhibi o s blocked Sos1/2-dependen gela in deg ada-
ion almos comple ely, independen o hei geno ype
(Fig.7B, C). These expe imen s suppo a model in which
he enhanced gela in deg ada ion obse ed in Sos1/2-
deple ed cells depends on hei inc eased exp ession o
Mmp2/9.
Pha macologic Sos1 inhibi ion does no ecapi ula e in
i o gene ically-media ed Sos1 abla ion in p ima y MEFs
Du ing ecen yea s, pha macological inhibi o s wi h
demons a ed abili y o di ec ly block Sos1::RAS in e -
ac ions ha e been de eloped [4]. In his ega d, BI-3406
has been p o en as a po en , selec i e and o ally a ail-
able, Sos1 inhibi o [24]. We hen aimed a e alua ing he
e ec o BI-3406 adminis a ion in WT p ima y MEFs.
We i s examined whe he pha macologic inhibi ion
o Sos1 caused mo phological al e a ions in he cell cy o-
a chi ec u e o p ima y MEFs. Ou esul s showed ha
BI-3406 adminis a ion in, WT MEFs, ba ely modi ied
cell mo phology, only exhibi ing a sligh inc ease in cell
a ea, wi hou a loss o he pola i y, o did no a ec o he
p o ein exp ession le els o phospho-Myosin in compa i-
son wi h he ehicle- ea ed coun e pa s (Fig.8A, D, E).
Cell coun measu emen in cul u es o BI-3406- ea ed
p ima y MEFs o he WT geno ype e ealed ha , pha -
macologically-media ed Sos1 inhibi ion did no impac
he cell g ow h o p ima y MEFs (Fig. 8B). We nex
examined he e ec o BI-3406 in he egula ion o cell
(See igu e on p e ious page.)
Fig. 3 Sos1 deple ion al e s ocal adhesion dynamics. (A) Rep esen a i e IRM images o indi idual p ima y MEFs om he ou indica ed geno ypes, aken
om imelapse mic oscopy mo ies. Boxes indica e egions shown in enla ged images in (B). Scale ba in B: 10μm. (C) Indi idual MEFs in A a e ep esen ed
wi h a colo code ( ed, g een and blue) ep esen ing he s abili y o FA h oughou he du a ion o he imelapse expe imen s (60min) scaling om low
(blue), medium (g een) and high ( ed) s abili y, espec i ely. (D) Cell sp ead a ea o MEFs om he ou geno ypes was e alua ed by IRM. Images we e gen-
e a ed by se ially o e lapping all mo ie images (1 image e e y 20s du ing 60min). (E) Images illus a e he cell pe ime e o indi idual MEFs a di e en
ime poin s using a colo code ha ep esen s he ela i e cell posi ion a he beginning o he expe imen ( ime 0, ed) a 30min (g een) and a 60min
(blue), espec i ely. In addi ion, whi e colo ep esen s con e gence zones and illus a es highly s able FA. Scale ba : 25μm. (F-I) Ba cha s ep esen he
numbe o FA pe cell (F) he ela i e pe cen age o in ensi y o FA (G), cell sp ead a ea (H) and he numbe o lamellipodia pe cell in MEFs o he ou
geno ypes. n = 12 cells pe geno ype ( om h ee expe imen al eplica es) (F and I), n = 9 pe geno ype ( om h ee expe imen al eplica es) (G) and n = 10
pe geno ype ( om h ee expe imen al eplica es) (H). Da a a e exp essed as mean ± SEM. S a is ical di e ences we e conside ed a : */# p < 0.05, **/##
p < 0.01 and ***/### p < 0.001 s. Sos1/2WT and Sos2KO, espec i ely
Page 16 o 18Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
exp ession o Mmp2/9 is cu en ly unknown and will
equi e u he analysis.
Finally, gi en he impo ance o he ecen ly epo ed
Sos1 inhibi o s as po en ial he apeu ic ools in ce ain
RAS-d i en malignancies [4, 25], we u he e alua ed
he e ec o he Sos1 inhibi o BI-3406 in di e en cel-
lula p ocesses in p ima y MEFs. Ou esul s demon-
s a ed ha , in con as o he esul s ob ained ollowing
gene ically-media ed Sos1 abla ion [18], BI-3406 ba ely
al e ed he cell a chi ec u e o p ima y MEFs and did no
impai cell p oli e a ion o cell mig a ion in his cell ype.
BI-3406 adminis a ion inc eased he capaci y o p ima y
MEFs o deg ade gela in, bu much less in compa ison
wi h he gene ic model o Sos1 emo al. O e all, hese
obse a ions sugges ha BI-3406 does no ecapi ula e
he e ec s obse ed when Sos1 is gene ically abla ed. In
his ega d, p io s udies e ealed ha BI-3406 p e e -
ably blocks in e ac ion o Sos1 wi h oncogenic Ras alleles
(mainly KRas), whe eas i shows no an ip oli e a i e
p ope ies in Ras WT umo cells [24], sugges ing ha
his compound p e e ably ac s o e oncogenic, KRas-
dependen , umo cells. In addi ion, whe eas BI-3406
has been p o en o bind o he ca aly ic domain o SOS1,
he eby p e en ing he in e ac ion wi h KRas, i has no
been e alua ed whe he i may in e e e Sos1-media ed
Rac ac i a ion. Ou wound-healing assays sugges he
BI-3406 p obably does no dis up Sos1-Rac in e ac ion.
Ne e heless, u he expe imen s a e needed, pa icu-
la ly in K as-mu an cells, in o de o e alua e whe he
hese small molecules could impac he capabili y o
umo cells o mig a e.
Conclusions
Ou p esen esul s add new, de ailed in o ma ion ega d-
ing he unc ional con ibu ion o Sos1/2, and especially
o Sos1, o he egula ion o cell mig a ion, which seem
o di e i analyzed unde 2D o 3D condi ions. In his
ega d, ou da a also un eiled a possible in ol emen o
Sos1/2 in he egula ion o Mmp2/9 in p ima y MEFs.
Abb e ia ions
CRIB Cdc42- and Rac-In e ac i e Binding mo i
DEG Di e en ially Exp essed Genes
DKO Double Knock-Ou
ECM Ex aCellula Ma ix
FAs Focal Adhesions
GDP Guanosine Diphospha e
GEF Guanine nucleo ide Exchange Fac o
GTP Guanosine T iphospha e
IRM In e e ence Re lec ion Mic oscopy
KO Knock-Ou
MEFs Mouse Emb yonic Fib oblas s
MMP Ma ix Me alloP o einase
PBS Phospha e Bu e ed Saline
SOS Son O Se enless
4OHT 4-Hyd oxyTamoxi en
WT Wild Type
Supplemen a y In o ma ion
The online e sion con ains supplemen a y ma e ial a ailable a h p s : / / d o i . o
g / 1 0 . 1 1 8 6 / s 1 2 9 6 4 - 0 2 5 - 0 2 1 2 2 - 1.
Supplemen a y Ma e ial 1
Au ho con ibu ions
Concep ion and Design: F.C.B, E.S, P.L.B, M.V-M and A.J.R. Da a acquisi ion,
analysis, and alida ion: P.L.B, R.G.-N, C. Ll, L.F.L.-M, R.F.-M, N.C, F.M.V, L.L-R, M.R.H,
A.J.R, M.V.-M, F.C.B. D a ing manusc ip : E.S, F.C.B. Re ising manusc ip : E.S,
F.C.B, P.L.B, R.G-N, L.F.L.-M, R.F.-M, F.M.V, A.J.R, X.R.B, M.V-M.
Funding
This wo k was unded by g an s ISCIII-MCUI (FIS PI19/00934), JCyL (SA264P18-
UIC 076), A eces Founda ion (CIVP19A5942) and ISCIII-CIBERONC (g oup
CB16/12/00352) o E.S; Soló zano-Ba uso Founda ion (FS/32-2020), Minis e io
de Ciencia e Inno ación (MICIU/AEI/ h p s : / / d o i . o g / 1 0 . 1 3 0 3 9 / 5 0 1 1 0 0 0 1 1 0 3
3 /, as pa o I + D + i P ojec “PID2022-136409OB-I00” and MICIU/AEI/ h p s : /
/ d o i . o g / 1 0 . 1 3 0 3 9 / 5 0 1 1 0 0 0 1 1 0 3 3 / and Nex Gene a ionEU/PRTR, as pa o
I + D + i P ojec “CNS2022-135292), Eugenio Rod iguez Pascual Founda ion
and Asociación Inés de Pablo Llo ens-GETTHI o F.C.B; Fede ación Española de
En e medades Ra as (AI-2023-015) o F.M.V; Minis e io de Ciencia e Inno ación
(PID2020-116232RB-I00) and ECRIN-M3 om AECC/AIRC/CRUK o M.V-M;
Cance Resea ch UK (C6620/A15961) o A.J.R. The X.R.B.’s p ojec leading o
hese esul s has ecei ed unding om he AECC (GC16173472GARC), he
Cas illa-León go e nmen (CSI018P23), g an s unded by MCIN/AEI/ h p s : / / d o i .
o g / 1 0 . 1 3 0 3 9 / 5 0 1 1 0 0 0 1 1 0 3 3 / plus he Eu opean Resea ch De elopmen Fund
«A way o making Eu ope» o he Eu opean Union (PID2021-122666OB-I00,
PDC2022-133027-I00, PLEC2022-009217), «la Caixa» Banking Founda ion
(HR20-00164), and he «Escale a de Excelencia» o he Educa ion Minis y o
he Cas illa y León au onomous go e nmen plus he Eu opean Resea ch
De elopmen Fund (CLU-2023-2-01). This esea ch was co- inanced by FEDER
unds. These CIC g oups a e suppo ed by he P og ama de Apoyo a Planes
Es a égicos de In es igación de Es uc u as de In es igación de Excelencia o
Cas illa y León au onomous go e nmen (CLC-2017-01) and AECC Excellence
p og am S op RAS Cance s (EPAEC222641CICS).
(See igu e on p e ious page.)
Fig. 8 The Sos1 inhibi o BI-3406 does no mimic he e ec s o gene ically-media ed Sos1 deple ion. (A) Rep esen a i e images o DMSO o BI-3406- ea -
ed WT p ima y MEFs o 24h aken om Incucy e. The g aphs show he mean cell a ea as well as he ci cula i y o MEFs in bo h expe imen al g oups.
Ci cula i y anges om 0 o 1, being 1 a pe ec ci cum e ence. Da a shown as mean ± SD. n = 3 independen expe imen s pe condi ion (50 cells/g oup).
* p < 0.05 s. ehicle- ea ed g oup. Scale Ba : 100μm. (B) G ow h cu e o ehicle- o BI-3406- ea ed (24h) p ima y MEFs in cul u e measu ed wi h Incu-
cy e. Da a shown as mean ± SD. n = 3 independen expe imen s pe condi ion. (C) Vehicle- (DMSO) o BI-3406- ea ed (1µM) con luen WT p ima y MEFs
we e sc a ched wi h a mic opipe e ip and closu e o he wounded a ea was eco ded o 24h by phase-con as pho omic oscopy (×10 magni ica ion).
A eas whe e cellula g ow h is no ye eco e ed a e he wound a e ma ked by su ounding yellow lines. Da a shown as mean ± SD. n = 3 independen
expe imen s pe condi ion. * p < 0.05 s. ehicle- ea ed g oup. (D) Vehicle- (DMSO) o BI-3406- ea ed (1µM) MEFs o he WT geno ype we e seeded on
gela in-FITC-coa ed co e slips o 8h, ixed and hen coun e s ained wi h phalloidin and DAPI. The ba cha illus a es he mean a ea o gela in deg ada-
ion (black a eas). Da a shown as mean ± SD. n = 3 independen expe imen s pe condi ion (a o al o 30 cells we e quan i a ed pe expe imen ). * p < 0.05
s. ehicle- ea ed coun e pa . (E) Wes e n blo s om p o ein ex ac s o Vehicle- o BI-3406-adminis e ed (24h) MEFs, o Mmp2 and phospho-Myosin
p o ein exp ession, using Tubulin as a con ol o no maliza ion,. Ba cha s illus a e he quan i a ion o he a io o Mmp2 and phospho-Myosin le els
ela i e o Tubulin. Da a shown as mean ± SD. n = 3 independen expe imen s pe condi ion
Page 17 o 18Lice as-Boillos e al. Cell Communica ion and Signaling (2025) 23:116
Da a a ailabili y
All mic oa ay aw da a ha e been uploaded and a e accessible a he NCBI
Gene Exp ession Omnibus (GEO) da abase (GSE277505).
Decla a ions
E hical app o al and consen o pa icipa e
All expe imen s we e app o ed by he Bioe hics Commi ee o he Cance
Resea ch Cen e (#417).
Compe ing in e es s
The au ho s decla e no compe ing in e es s.
Au ho de ails
1Lab 1, Cen o de In es igación del Cánce - IBMCC (CSIC-USAL) and
CIBERONC, Uni e sidad de Salamanca, Salamanca 37007, Spain
2Molecula Mechanisms P og am, Cen o de In es igación del Cánce ,
Ins i u o de Biología Molecula y Celula del Cánce , Consejo Supe io de
In es igaciones Cien í icas (CSIC) and Uni e si y o Salamanca,
Salamanca 37007, Spain
3Ecole Poly echnique édé ale de Lausanne, Lausanne, Swi ze land
4Depa amen o de Fisiología Medica y Bio ísica, Facul ad de Medicina,
Uni e sidad de Se illa and Ins i u o de Biomedicina de Se illa (IBiS)
(Hospi al Uni e si a io Vi gen del Rocío, CSIC/Uni e sidad de Se illa),
Se illa 41013, Spain
5Depa amen o de Biología Celula , Facul ad de Biología, Uni e sidad de
Se illa and Ins i u o de Biomedicina de Se illa (IBiS) (Hospi al Uni e si a io
Vi gen del Rocío, CSIC/Uni e sidad de Se illa), Se illa 41012, Spain
6Randall Cen e o Cell and Molecula Biophysics, King’s College London,
Guy’s Campus, New Hun ’s House, London SE1 1UL, UK
7School o Cellula and Molecula Medicine, Biomedical Sciences Building,
Uni e si y Walk, Uni e si y o B is ol, B is ol BS8 1TD, UK
8Lab 2, Cen o de In es igación del Cánce - IBMCC (CSIC-USAL) and
CIBERONC, Uni e sidad de Salamanca, Salamanca 37007, Spain
Recei ed: 24 Oc obe 2024 / Accep ed: 23 Feb ua y 2025
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Publishe ’s no e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in
published maps and ins i u ional a ilia ions.
