Fed Up With TheRucaparibNews Reports? Our Company Is On This Site For Your Needs

ing CycIF with live-cell imaging. CycIF is often effec-
resultsinhigherlevelsofcellloss(althoughnotallantibodiesare Tired Of AllRucaparibChit Chat? I'm At This Website Available For You tively mixed with live-cell imaging. Within the four-cycle, 9-
compatible with paraformaldehyde). While in the situation of protease- channel experiment proven in Fig. 2a, we monitored nuclear
mediated antibody inactivation (protocol variant 2), the combine of translocationofFoxO3ainMCF10AcellsstablyexpressingYFP-
protease and digestion time has to be optimized for each set of FoxO3aandmCherry-NLSfor24hfollowedby?xationandfour
samples to stability antibody inactivation with retention of cell cyclesof?uorophoreinactivationandantibodystaining.Weused
S807/S811
numberandimmunogenicity(ingeneral,papainandpepsinwork conjugated antibodies to measure cell cycle state (p-Rb
,
withmostcelllines;SupplementaryFig.

3).
p21,Ki-67andPCNA;seeSupplementaryTables1and2forthe
a
Dwell cell imaging of YFP-FoxO3a translocation
CycIF Sick And Tired Of So ManyRucaparibStories? Our Company Is At This Website For You (4 cycles)
1st
Slice = 150
Slice = 204
Slice = 244
2nd
10 ?M
N/C = 0.3672
1.6
N/C = 1.3484
N/C = 0.4688
ten?M
FoxO3a
p-Rb
DNA
PCNA
p21
DNA
3rd
4th
1.2
0.8
0.4
0
ten?M
10 ?M
150
165
180
Time slice (6 min per slice)
195
210
225
240
EGFR
Tubulin
DNA
Ki-67
p-S6
S235/6
DNA
b
CycIF composite
Morphometry
Microtubules
Fiber length
branching pattern
mitotic structures and so forth.
EGFR
Cluster amount
cluster intensity
internalization and so on.


50 ?M
EGFR
VEGFR2
Tubulin
DNA
c
Clustering morphometric functions
Cluster 1
(S-phase)
Cluster 2
(Other)
Vimentin common length
Microtubule count
VEGFR2 count
EGFR count
Vimentin count
Vimentin coverage (percent)
Microtubule typical length
Microtubule coverage (%)
EGFR feret angle
EGFR feret
EGFR common dimension
VEGFR2 typical size
VEGFR2 feret
VEGFR2 feret angle
PCNA Tired Of So ManyBirinapantScoops? We're On This Website To Meet Your Requirements! foci count
VEGFR2
PCNA
PCNA foci regular size
Figure2|CompatibilityofCycIFwithlive-cellimaging,analysisofsubcellularlocalizationofproteinsandmorphometricfeatures.(a)ApplyingCycIFat
theendofFP-basedlive-cellimaging.NucleartranslocationofFoxO3ainMCF10Acells(stablyexpressingYFP-FoxO3aandmCherry-NLS)wasmonitored
for24h(leftpanel).RedarrowsindicateFoxO3anucleartocytoplasmic(N/C)ratiosattwosnapshots.Cellswerethen?xedandsubjectedtofour-cycle
CycIFusing?uorophore-conjugatedantibodiesp-RbS807/S811(cycle1),p21andPCNA(cycle2),EGFRandb-tubulin(cycle3)andKi-67andp-S6S235/6
(cycle4)(rightpanel).

(b)MorphometricanalysisofCycIF-generatedimagesofsinglecells.ThreeroundsofCycIFstainingwereperformedasindicatedin
SupplementaryFig.S8.Therawintensity-basedimagesofEGFR,VEGFR2,Tubulin,VimentinandPCNAwerethenbinarizedandpassedthroughdifferent
?lters(sharpen,skeletonizedandmaximum,andsoon)forextractingtexturefeatures(length,branches,enrichmentandclusters).Tworepresentative
images/masksfromTubulin/microtubuleandEGFRareshownhere.Detailedmethodsandotherimages/maskscouldbefoundinSupplementary
Details.(c)Single-cellclusteringbasedonmorphometricfeatures.Sixteendifferentfeatureswereextractedfrom?vedifferentIFsignals.Thenumerical
valuesfromfeatureswereusedinhierarchicalclusteringofsinglecells(leftpanel).Twosub-clustersweredisplayedwithdistinctfeaturesgeneratedfrom
PCNAandVEGFR2(rightpanel).Thehierarch