A magnetic switch for the control of cell death signalling i(3)

culture slide containing DLD-1cells by using the magnetic set-up shown in Fig.4a.On application of the magnetic field for 4h,activation of caspase-3,seen in the form of an immunostained red fluorescence signal,is observed to occur exclusively in the area of about 200µm ×100µm between the magnets (Fig.4b).Similarly,active caspase-3is consistently observed in an array comprising magnetic focusing on three local spots (Fig.4c,d),which testifies to the spatial controllability of the magnetic switching technique.

Although this magnetic switching strategy for apoptosis signalling is effective at the in vitro level,its further extension to in vivo activation is both significant and challenging because of the complexity of live biological systems.We choose zebrafish as an in vivo model not only because of the benefits associated with optical imaging and convenient screening 32–34,but also because of the genetic closeness of the zebrafish ovarian TNF receptor (OTR)to the human DR4for apoptosis signalling 35,36.Zebrafish OTRs are targeted and magnetically manipulated by using zebrafish OTR antibody conjugated to MNPs (zAb–MNPs).First,fluorescein-labelled zAb–MNPs (2.5ng per embryo)are micro-injected into the yolk of the zebrafish embryo at the one-cell stage of growth.After 24h,pronase is used to hatch the zebrafish embryos,which are then divided into two groups including those that will and will not be magnetically activated.For the magnetically activated group,a magnetic field with a strength of

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Fluorescein- conjugated B r i g h t -f i e l d i m a g e

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Figure 5|In vivo magnetic apoptosis signalling for zebra?sh.a ,The apoptosis experimental scheme of zebra?sh.Fluorescein-labelled zAb–MNPs

(2.5ng embryo ?1)are micro-injected into yolk of embryo at one-cell stage to label extrinsic apoptosis receptor (OTR).At 24h post-fertilization (h.p.f.),the magnetic ?eld is applied to zebra?sh.b ,Bright-?eld microscope images of three different groups of zebra?sh,control (i),non-activated (ii)and

magnetically activated (iii).The magnetically activated group of zebra?sh shows morphological alterations in the tail region compared with other groups.c ,Quantitative analysis on tail bending by measuring the angle between the line on the pronephros (PR)and the line of tail tip (TT)for each group.

d –f ,Fluorescenc

e images o

f zebra?sh in which zAb–MNPs are green and active caspase-3is immunostained as red.Green and red ?uorescence are only observed in the tail region of the magnetically activated group (d (iii),e (iii)).Insets in d and e are magni?ed images of tail region.f ,Magni?ed ?uorescence micrographs of active caspase-3in zebra?sh tail region.

g ,A grap

h of caspase-3activity measured by using ?uorescence intensity of zebra?sh.Error bars are standard deviation.(???P <0.001).

0.50T is applied to a zebrafish chamber for 24h (Fig.5a).After application of the magnetic field,characteristic features associated with apoptosis,such as morphological deformation of embryos and caspase-3activation 35,36,are examined at the 48h post-fertilization (h.p.f.)stage.Inspection of bright-field microscope images shows that both the control and non-activated groups exhibit normal ontogenic zebrafish embryo development (Fig.5b(i,ii)).In contrast,morphological alterations in the tail region are clearly observed in images of the magnetically activated group (Fig.5b(iii)).This morphological change is visualized by determining the angle of tail tip bending.For this purpose,a straight line is drawn along the pronephros and the angles between this line and tail tip line are measured in three different groups,as shown in Fig.5c.The magnetically activated group shows an approximately 3.5-fold larger angle (about 22?)than that of the control and non-activated groups (about 6?).This morphological change is proposed to be a consequence of apoptosis signalling 35.Besides,zebrafish embryos

without zAb–MNPs injection are not affected by magnetic field application alone (Supplementary Section S7).

To gain further evidence for the occurrence of apoptosis,the location of zAb–MNPs and the apoptosis signalling products are examined using an optical method.zAb–MNPs are fluorescently labelled and the strong green fluorescence signal is observed in the yolk for both the magnetically activated and non-activated groups owing to residual zAb–MNPs after the injection.Interestingly,clumps of strong green fluorescence signal are seen in zebrafish tail regions that are magnetically activated,whereas this region of non-activated zebrafish shows only a dispersed and weak green fluorescence signal (Fig.5d(ii,iii)).These observations are caused by the combination of higher OTR expression in the tail region of zebrafish in a manner that is consistent with previous observations 35,36,and subsequent magnetic clustering of the OTRs.The activation of caspase-3at the tail region is confirmed by using immunostaining.Both the control and non-activated

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