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Necrotic pyknosis is a morphologically and biochemically distinctevent from apoptotic pyknosisLin Hou1,2, Kai Liu1, Yuhong Li2, Shuang Ma2, Xunming Ji2,* and Lei Liu2,*

ABSTRACTClassification of apoptosis and necrosis bymorphological differenceshas been widely used for decades. However, this usefulness of thismethod has been seriously questioned in recent years, mainly dueto a lack of functional and biochemical evidence to interpret themorphology changes. To address this matter, we devised geneticmanipulations in Drosophila to study pyknosis, a process of nuclearshrinkage and chromatin condensation that occurs in apoptosis andnecrosis. By following the progression of necrotic pyknosis, wesurprisingly observed a transient state of chromatin detachment fromthe nuclear envelope, followed by the nuclear envelope completelycollapsing onto chromatin. This phenomenon led us to discover thatphosphorylation of barrier-to-autointegration factor (BAF) mediatesthis initial separation of nuclear envelope from chromatin.Functionally, inhibition of BAF phosphorylation suppressed necrosisin both Drosophila and human cells, suggesting that necroticpyknosis is conserved in the propagation of necrosis. In contrast,during apoptotic pyknosis the chromatin did not detach from thenuclear envelope and inhibition of BAF phosphorylation had no effecton apoptotic pyknosis and apoptosis. Our research provides thefirst genetic evidence supporting a morphological classification ofapoptosis and necrosis through different forms of pyknosis.

KEY WORDS: BAF phosphorylation, Drosophila genetics,Apoptosis, Cell death morphology, Necrosis, Pyknosis

INTRODUCTIONMorphological differences observed during cell death have beenwidely used to classify apoptosis and necrosis for a long time. Theseinclude apoptotic features such as cell shrinkage, membraneblebbing, nuclear condensation and apoptotic body formation(Kerr et al., 1972), and necrotic features such as cell swelling,plasma membrane rupture, intracellular vacuolization and nuclearchromatin clumping (Raffray and Cohen, 1997). However, manyexceptions have been discovered and classification of cell death bymorphology has been controversial (Raffray and Cohen, 1997).Recently, the Nomenclature Committee on Cell Death (NCCD) hasrecommended using biochemical markers for cell deathclassification instead of morphology (Galluzzi et al., 2015, 2012).The reasons include that morphology might not be linked to afunctional aspect of cell death, a given morphology might be

triggered by heterogeneous insults, and intermediate morphologyshared by both apoptosis and necrosis might exist (Galluzzi et al.,2015, 2012; Raffray and Cohen, 1997).

Pyknosis has been considered as an irreversible condensationof chromatin and the nucleus. It commonly occurs in bothapoptotic and necrotic cell death. For apoptosis, the nucleususually undergoes condensation, chromatin marginalization andfragmentation into a few large and regular chromatin clumps, whichare eventually packed into apoptotic bodies (Kerr et al., 1972;Niquet et al., 2003). In contrast, nuclei in necrotic cells condenseinto to smaller chromatin clumps with irregular and dispersedmorphologies, which might be dissolved later (Bortul et al., 2001;Fujikawa et al., 2000; Hardingham et al., 2002; Niquet et al., 2003).Therefore, based on the morphology of nuclear fragmentation,pyknosis can be divided into nucleolytic pyknosis (mainlyoccurring in apoptosis) and anucleolytic pyknosis (mainlyoccurring in necrosis) (Burgoyne, 1999). Although pyknosis hasbeen widely considered as a marker of cell death in vitro and in vivo(Colbourne et al., 1999; Fujikawa et al., 1999, 2010; Ji et al., 2013;Niquet et al., 2003; Sohn et al., 1998), it is unclear whether pyknosisis a regulated process.

Here, we studied the morphological changes visible in pyknosisduring the execution of apoptosis and necrosis in a temporalmanner. We found that necrotic pyknosis occurred in a distinctpattern compared to apoptotic pyknosis, and phosphorylation ofBAF plays a key role only in necrotic pyknosis.

RESULTS AND DISCUSSIONNecrotic pyknosis shows a different morphology toapoptotic pyknosisTo study necrotic pyknosis, we used a previously establishednecrosis model in Drosophila. By allowing transient expression ofa leaky cation channel, the glutamate receptor 1 Lurcher mutant(GluR1Lc), Ca2+ is overloaded into cells, which results in necrosisand fly lethality (Liu et al., 2014). This fly model contains the Gal4driver Appl-Gal4 (expressed in neurons and a few epithelial cells inthe larval anal pad), and the UAS expression constructs UAS-GluR1Lc and tub-Gal80ts, and we denote it the AG model(Appl>GluR1Lc, tub-Gal80ts) (Liu et al., 2014). The AG flies arehealthy at 18°C due to inhibition of Gal4 function by Gal80ts. Uponshifting flies to 30°C, Gal80ts is deactivated and necrosis is initiatedby GluR1Lc expression (Liu et al., 2014). To label the nuclearenvelope and the chromatin, the fluorescent reporters UAS-koi.GFPand His2Av-mRFP1 (expressing His2Av–mRFP fusion protein inall cells) were used, respectively.

After being at 30°C for 22 h, the nuclei of control flies [Appl-Gal4;tub-Gal80ts, labeled as wild type (WT); Fig. 1Aa] showednormal morphology, with chromatin occupying the whole nucleus,and the interaction of chromatin with the nuclear envelope wasclearly visible. However, after 18 to 20 h at 30°C, the chromatin inthe epithelial cells of AG flies was dramatically condensed, whereasReceived 14 December 2015; Accepted 23 June 2016

1State Key Laboratory of Membrane Biology, School of Life Sciences, PekingUniversity, Beijing 100871, China. 2Aging and Disease Laboratory of XuanwuHospital and Center of Stroke, Beijing Institute for Brain Disorders, Capital MedicalUniversity, Youanmen, Beijing 100069, China.

*Authors for correspondence ( jixm@ccmu.edu.cn; leiliu@ccmu.edu.cn)

K.L., 0000-0002-0137-7657; K.L., 0000-0002-6387-7745; X.J., 0000-0002-0527-2852; L.L., 0000-0002-6387-7745

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mailto:jixm@ccmu.edu.cnmailto:leiliu@ccmu.edu.cnhttp://orcid.org/0000-0002-0137-7657http://orcid.org/0000-0002-6387-7745http://orcid.org/0000-0002-0527-2852http://orcid.org/0000-0002-0527-2852http://orcid.org/0000-0002-6387-7745

Fig. 1. Characterization of pyknosis in Drosophila. (A) Observation of necrotic pyknosis in vivo in larval anal pad epithelial cells. The nuclear envelope andchromatin were labeled byUAS-koi.GFP andH2Av-mRFP1, respectively. (a) The nuclear envelope and chromatin in wild-type (WT) cells (Appl-Gal4;tub-Gal80ts)kept at 30°C for 22 h. (b,c) The early stage of nuclear envelope and chromatin changes in AG flies (Appl-Gal4;tub-Gal80ts, UAS-GluR1Lc/cyo) kept at 30°C for theindicated time. (d) The late stage of nuclear envelope and chromatin changes in AG flies after 22 h at 30°C. Representative images from n=10 experiments.For each experiment, 50 cells were observed. (B) Observation of apoptotic pyknosis in vivo in larval anal pad epithelial cells from AR flies kept at 30°C for theindicated time. The nuclear envelope and chromatin were labeled by UAS-koi.GFP and H2Av-mRFP1, respectively. (C) BAF protein localization in vivo. Themicrographs show the pattern of GFP-tagged wild-type BAF (GFP–BAF-WT) under wild-type (a), AG (b) andAR (c) backgrounds in larval anal pad epithelial cellskept at 30°C for 18 h. In addition, the patterns of GFP–BAF-3A (d) andGFP–BAF-3D (e) under theAG background are shown. (D) List of potential phosphorylationsites in the N-terminus of BAF. Red letters highlight the mutations studied in this paper. (E) An example of the effect of the BAF-3A mutant protein on the early-stage of necrotic pyknosis (disassociation of chromatin from nuclear envelope) in the indicated lines and conditions. (F) Quantification of data from the experimentshown in E. The bar graph shows the percentage of normal nuclei without nuclear envelope and chromatin disassociation. n=5 experiments with dataquantified from four (WT), seven (w1118) and seven (BAF-3A) larvae in each experiment. (G) Effect of BAF-3A on late-stage of necrotic and apoptoticpyknosis in larval anal pad epithelial cells in the indicated lines and conditions. Chromatin changes were revealed by H2Av–mRFP1. White dotted lines markthe boundary of the anal pad. (H) Quantification of data from the experiment shown in G. The bar graph shows the number of fragmented, anucleolyticpyknotic or normal nuclei in the larval anal pad. n=3 experiments with data quantified from five larvae in each experiment. Data in all bar graphs are means+s.d.**P

the nuclear envelope only slightly shrank, leading to the detachmentof chromatin from the nuclear envelope (Fig. 1Ab,c). Later, both thenuclear envelope and the chromatin were further compactedand eventually collapsed together (Fig. 1Ad). The morphology isconsistent with the features of anucleolytic pyknosis (Fujikawaet al., 2010; Sohn et al., 1998).We also studied apoptotic pyknosis in vivo by transient

expression of reaper (rpr) (Appl>rpr, tub-Gal80ts, denoted AR),which induces classical apoptosis (White et al., 1996). We foundthat the nuclear envelope and chromatin physically shrank together,with the nucleus eventually fragmenting into regular clumps(Fig. 1Ba–d).

Phosphorylation of BAF specifically mediates necroticpyknosisOur data suggest that necrotic pyknosis is likely to be initiated by thedetachment of chromatin from the nuclear envelope. Previousstudies have shown that barrier-to-autointegration factor (BAF)plays a key role in chromatin tethering to the nuclear envelopethrough its interaction with LAP2, emerin and MAN1 (LEM)-domain-containing proteins and double-stranded DNA (dsDNA) ina sequence non-specific manner (Umland et al., 2000; Zheng et al.,2000).Based on the function of BAF, it is possible that the dissociation

of chromatin from the nuclear envelope is induced by deregulationof BAF during necrosis. To examine BAF localization in necrosis,we generated a GFP-tagged BAF transgene (UAS-GFP-BAF). It hasbeen reported that an N-terminal GFP tag does not affect BAFactivity (Nichols et al., 2006; Shimi et al., 2004). In wild-type cells(Appl-Gal4;tub-Gal80ts) after 30°C for 18 h, GFP–BAF wasdistributed on chromatin and interacted with the nuclear envelope(Fig. 1Ca). In the AG flies, most GFP–BAF protein was alsolocalized on the compacted chromatin (Fig. 1Cb), suggesting thatBAF localization is not drastically altered during necrosis. However,GFP–BAF reduced its distribution in the chromatin and formedring-like structures in apoptosis (Fig. 1Cc). This phenomenon isconsistent with a previous report stating that BAF disassembled anddisappeared from the nucleus during apoptosis (Furukawa et al.,2007).The N-terminal phosphorylation of BAF has been reported to

cause the detachment of chromatin from the nuclear envelopeduring karyosome formation in the meiosis of the oocyte inDrosophila (Lancaster et al., 2007). The BAF N-terminus has threepotential phosphorylation sites, including serine 2, threonine 4 andserine 5. To test the importance of BAF phosphorylation in necroticpyknosis, we generated transgenic flies expressing BAF proteinswith these three potential phosphorylation sites mutated to alanine(the non-phosphorylatable form, denoted BAF-3A) or aspartic acid(the phospho-mimic form, denoted BAF-3D). These mutation sitesof BAF are illustrated in Fig. 1D. When these mutant flies werecrossed to AG flies to induce necrosis, GFP–BAF-3A localizationappeared to be similar to the pattern of wild-type BAF (GFP–BAF-WT) under normal conditions (Fig. 1Cd), whereas GFP–BAF-3Dshowed a similar dissociation phenotype to that of the GFP–BAF-WT in the AG background (Fig. 1Ce). These data suggest thatchromatin disassociation from nuclear envelope during necrosis islikely inhibited by GFP–BAF-3A expression. To further confirmthe role of BAF-3A, we quantified the number of cells with earlystage necrotic pyknosis features in cells with the chromatin andnuclear envelope labeled by His2Av–mRFP1 and koi.GFP. In theAG flies, 58% of cells displayed the early morphological features ofnecrotic pyknosis (Fig. 1Ea,b,F). Strikingly, the early stage

morphology defect of necrotic pyknosis was completely abolishedby the BAF-3A expression (Fig. 1Ec,F). Similarly, expression ofBAF-3A rescued the late-stage morphology of necrotic pyknosis(Fig. 1Ga–c,H). For apoptotic pyknosis, BAF-3A expression had noinhibitory effect (Fig. 1Gd,e,H), indicating different molecule(s)might be required.

Anucleolytic pyknosis plays a functional role in necrotic celldeathAlthough anucleolytic pyknosis has been considered to be a markerof necrosis, its function in cell death is unclear. To address thisquestion, we investigated the survival of AG flies under differenttransgenic BAF mutant backgrounds. Transient overexpression ofBAF-3A or BAF-3D in neurons had no effect on fly survival(Fig. 2A). Upon induction of necrosis, the survival rate of adult AGflies decreased to 38.4% (Fig. 2A). Overexpression of BAF-WTresulted in a similar survival rate (Fig. 2A). Strikingly, theoverexpression of BAF-3A increased the survival rate to 70.0%,whereas the expression of BAF-3D reduced the survival rate to11.7% (Fig. 2A). At the cellular level, BAF-3A suppressed thenecrotic morphology, whereas BAF-3D enhanced it, and BAF-WThad no effect (Fig. 2B,C). Taken together, BAF phosphorylationalone is not sufficient to induce cell death, but it is necessary fornecrotic pyknosis and the propagation of cell death. In contrast, theexpression of BAF-3A or BAF-3D had no effect on fly models ofapoptosis, including the eye defects of GMR-Hid (Grether et al.,1995) andGMR>eiger (Hid and Eiger induce caspase-mediated andJNK-mediated apoptosis, respectively; Moreno et al., 2002)(Fig. 2D).

Owing to a lack of antibody that can directly detect thephosphorylation of Drosophila BAF, we assessed BAFphosphorylation by immunoprecipitating GFP–BAF proteins withan anti-GFP antibody followed by western blotting with an antibodyagainst phosphorylated threonine (pThr). The result showed thatBAF phosphorylation indeed increased in the AG flies, and theexpression of BAF-3A abolished the phosphorylation (Fig. 2E). Thesubtle change in BAF phosphorylation is likely due to only ∼1% ofneurons undergoing necrosis, which is sufficient to cause flylethality in the AG flies (Liu et al., 2014). No signal could bedetected when an antibody against phosphorylated serine wasapplied. This result suggests that threonine 4 in the N-terminal ofBAF is the functional site that is phosphorylated during necroticpyknosis. To test this hypothesis, we generated transgenes to expressBAF proteins with both threonine 4 and serine 5 mutated to alanine(BAF-2A) or threonine 4 alone mutated to alanine (BAF-1A)(Fig. 1D). We found that both BAF-2A and BAF-1A could rescuethe lethality of the AG flies, similar to BAF-3A (Fig. 2A).

BAF phosphorylation regulates necrotic pyknosis inmammalian cellsBecause of the conserved function of BAF in chromatin anchoringin metazoans (Segura-Totten andWilson, 2004), we asked whetherBAF phosphorylation regulates necrotic pyknosis in mammaliancells. To induce necrosis, human neuroblastoma SH-SY5Y cellswere treated with Ca2+ ionophore to overload Ca2+. Ca2+ ionophoretreatment led to nuclear shrinkage into propidium-iodide-positive,small, round and bright chromatin clumps within 30 min(Fig. 3Aa–b′). However, we did not observe the transientdetachment of chromatin from the nuclear envelope in SH-SY5Ycells and in several other cultured mammalian cells (Fig. 3Ac;Fig. S1). This variation might be due to the difference betweenin vivo and in vitro systems. In tissues, the cellular contacts play an

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important role in setting the threshold of cell death (Raffray andCohen, 1997). In fact, the intermediate state of the nuclearenvelope being detached from chromatin has been observed in theretinoblastoma patients (Buchi et al., 1994). In mammalian cells,

the nucleus is highly compacted and the dissociation of nuclearenvelope from chromatin might be less obvious at the cellmorphological level. To study the interaction between thenuclear envelope and chromatin in mammalian cells, the DNA

Fig. 2. Functional role ofBAFphosphorylationonnecrosis andapoptosis. (A) Theeffect of expression of BAFmutants on the survival ofAG flies. The flies wereincubated at 30°C for 12 h. Then, the flies were returned to 18°C, and the fly survival was recorded 48 h later. n=3 experiments for the control flies (Appl-Gal4; Tub-Gal80ts) and n=7 for the AG flies. Fifty flieswere tested for each experiment. (B) The effect of the BAFmutants on necrosis in larval anal pads. Larvaewere incubatedat 30°C, for 22h, 26h and 20h (top, middle and bottom panels, respectively). A later time point (26 h) is shown to demonstrate the rescue effect of BAF-3A, and anearlier time point (20 h) is shown to demonstrate the enhanced effect of BAF-3D. A representative image from four or five experiments is shown. (C) Quantification ofcell number for the experiments shown in B. n=6 (BAF-WT), n=7 (BAF-3A) and n=7 (BAF-3D) larvae. (D) Effects of BAF mutants on the caspase-dependentapoptosis and the JNK-dependent cell death models inDrosophila eyes. Overexpression of IAP1 and bskDN are shown as the positive controls, which suppress theindicated cell death pathways. (E) Immunoprecipitation (IP) followed by immunoblotting (WB) to detect BAF phosphorylation at threonine residues (pThr) duringnecrosis. A representative experiment from n=3 is shown. The asterisk indicates a non-specific band present in all samples, which runs slightly higher than thephosphorylated GFP–BAF band (

Fig. 3. Characterization of necrotic pyknosis in the human SH-SY5Y cells. (A) The change of nuclear morphology upon the induction of necrosis. (a,a′ andb,b′) DAPI and propidium iodide (PI) staining of the same human SH-SY5Y cells. In b,b′ and c, the cell cultures were treated with Ca2+ ionophore (20 mM) for30 min. b and b′ show the chromatin condensation (DAPI) in necrotic cells (propidium iodide positive). (c) Co-staining of chromatin (DAPI) and the nuclearenvelope (anti-NPC antibody) in a necrotic cell. (B) Quantification of lamin-B1-bound DNA in human SH-SY5Y cells expressing the indicated form of BANF1, asassessed by agarose gel electrophoresis. Results are means±s.d. relative to the level of the control (IgG precipitation without adding Ca2+ ionophore) which wasset at 1. n=3 experiments; a representative gel is also shown. (C) Quantification of the lamin-B1-bound DNA by qPCR. For each sample, the Ct value of thechromatin immunoprecipitation (chIP) DNA fraction was normalized to its own input DNA fraction. The DNA level of the normalized background (IgG chIP) was setas 1. n=3 experiments; for each experiment, the MALBAC and qPCR were performed twice. (D) Comparison of the N-terminal sequences of Drosophila BAF(dBAF) and human BANF1 (hBANF1). The potential phosphorylation sites are indicated by blue arrowheads. Red letters indicate non-phosphorylatable mutationsites. (E) Effect of BANF1 on necrosis. Stable human SH-SY5Y cell lines expressing GFP (a,b), wild-type BANF1 (BANF1-WT) (c) or non-phosphorylatableBANF1 (BANF1-3A) (d) were treated with DMSO or Ca2+ ionophore for 30 min and necrotic cells are revealed by propidium iodide staining. The upper and lowerpanels are the same views with DAPI and propidium iodide staining, respectively. (F) Quantification of results shown in E. n=3 experiments; data were quantifiedfrom six (for the control GFP+DMSO) and eight images (for treatment with Ca2+ ionophore). (G) Cell viability quantified by an ATP assay. n=4. (H)Immunoprecipitation (IP) followed by immunoblotting (IB) to detect BANF1 phosphorylation during necrosis. A representative image from n=3 is shown. *P

that tethered on the inner nuclear membrane was precipitated byan anti-lamin-B1 antibody. Then, the precipitated DNA wasquantified by an agarose gel and a quantitative PCR (qPCR) assay.The result showed that the amount of lamin-B1-bound DNAwas significantly decreased during necrosis in the SY5Y cells(Fig. 3B,C). This result indicates that the dissociation of chromatinand nuclear envelope might also take place during necrosis in themammalian cells.The potential phosphorylation sites of human BAF (BANF1)

include threonine 2, threonine 3 and serine 4 (Fig. 3D). Because thesite equivalent to the Drosophila BAF threonine 4 was unclear(Lancaster et al., 2007), we mutated all three sites (Fig. 3D, BANF1-3A), and expressed the mutant constructs in SH-SY5Ycells. The result showed that expression of BANF1-3A (a non-phosphorylatable mutant) abolished the reduction of lamin-B1-bound DNA during necrosis (Fig. 3B,C). To examine the functionalrole of BANF1 phosphorylation on necrosis, we performed Ca2+

ionophore experiments. Ca2+ ionophore treatment induced ∼30.4%propidium-iodide-positive nuclei (Fig. 3E,F). However, expressionof BANF1-3A reduced the proportion of cells that died to 14.4%(Fig. 3Ec,d,F). BANF1-3A also blocked the ATP depletion uponCa2+ ionophore treatment (Fig. 3G).To examine BANF1 phosphorylation, BANF1–Flag proteins were

precipitatedwith an anti-Flag antibodyand assessedwith an anti-pThrantibody. The result showed that the phosphorylation of BANF1-WT–Flag was not detectable under normal conditions but it wasgreatly increased upon treatment with Ca2+ ionophore (Fig. 3H).Importantly, there was no detectable phosphorylation of BANF1whenBANF1-3A–Flagwas expressed (Fig. 3H). This result indicatesthat BAF phosphorylation is a conserved event of necrosis.Our study identifies BAF phosphorylation as a specific

biochemical marker for necrotic pyknosis in certain types of cells,suggesting that classical apoptotic and necrotic pyknosis might bedistinct processes at the molecular level. A schematic model ofnecrotic pyknosis is shown in Fig. 4. For classification of cell death,identification of more biochemical markers that directly regulatemorphology should greatly improve the uncertainty in categorizingmore complicated modes of cell death (Raffray and Cohen, 1997).In fact, several regulators of apoptosis and necrosis have beenidentified, including caspase-activated DNase (CAD, also known as

DFFB), endonuclease G and DNase I in nuclear fragmentation(Enari et al., 1998; Li et al., 2001; Liu et al., 1997; Oliveri et al.,2001), and phospholipase A2 in necrotic pyknosis (Shinzawa andTsujimoto, 2003). Here, we provide an example of morphologicalregulation of necrotic pyknosis by a biochemical event.

MATERIALS AND METHODSDrosophila stocks and maintenanceFlies were raised on standard cornmeal medium. The stocks were kindlyprovided by colleagues including: UAS-eiger (Lei Xue, School of LifeScience and Technology, Tongji University, China), GMR-hid, GMR-Gal4(Andreas Bergmann, Department of Cancer Biology, University ofMassachusetts Medical School, Worcester, MA) and UAS-IAP1 (DeniseMontell, Molecular, Cellular, and Developmental Biology, University ofCalifornia, Santa Barbara, CA). We generated the following lines from thew1118 background by P-element insertion: UAS-GFP-BAF-WT, UAS-GFP-BAF-3A, UAS-GFP-BAF-3D, UAS-BAF-WT-Flag, UAS-BAF-3A-Flag,UAS-BAF-3D-Flag, UAS-GFP-BAF-2A and UAS-GFP-BAF-1A. UAS-koi.GFP (BL#26266) and H2Av-mRFP1 (BL#23650) were obtained from theBloomington Drosophila Stock Center.

Protein extraction and immunoprecipitationAll cell lines were obtained from the China Infrastructure of Cell LineResources and tested for contamination. Fly heads and human SH-SY5Ycells treated with Ca2+ ionophore (A23187; Tocris Bioscience #1234) werelysed in buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5% NP40)supplemented with protease inhibitor cocktail (Roche) and phosphataseinhibitors (1 mM Na3VO4, 50 mM NaF, 30 mM glycerophosphate and0.5 mM EDTA). After sonication and centrifugation, the supernatant wasincubated with normal IgG (1:1000, Santa Cruz Biotechnology, sc-2025)and protein-A/G–agarose beads (Thermo Scientific Pierce, #20421). Aftercentrifugation, the supernatant was incubated with anti-GFP antibody(1:200, Abcam ab1218, clone 9F9.F9) or anti-Flag antibody (1:1000,Sigma-Aldrich F3165, clone M2) for 2 h. Then, pre-washed beads wereadded and incubated overnight. After washing with lysis buffer six times,the pellet was boiled in SDS loading buffer. Tricine-SDS-PAGEwas used toseparate the small-molecular-mass proteins. BAF phosphorylation wasdetected with an anti-pThr antibody (1:500, Cell Signaling #9381).

Nuclear morphology and cell survival in cultured cellsAntibody against the nuclear pore complex (NPC) (1:1000, Abcamab24609, clone Mab414) was used for immunostaining. SH-SY5Y cellswere incubated with 20 mM Ca2+ ionophore for the indicated times. The

Fig. 4. A schematic model for necrotic pyknosis.At the early stages of necrotic pyknosis, BAFphosphorylation promotes condensed chromatin todissociate from the nuclear envelope. Then, at laterstages, the nuclear envelope collapses onto thechromatin and the plasma membrane is damaged.BAF-ph, phosphorylated BAF.

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ATP level was determined by a CellTiter-Glo® Luminescent Cell ViabilityAssay kit (Promega). The relative ATP level (%)=ATP [with Ca2+

ionophore]/ATP [without Ca2+ ionophore]×100.

Chromatin immunoprecipitation and quantitative PCRAfter Ca2+ ionophore or DMSO treatment for 30 min, chromatinimmunoprecipitation was performed as described previously (Boyd et al.,1998). Protein A+G agarose, salmon sperm DNA (Merck Millipore#16-201) and anti-lamin-B1 antibody (1:100, ab16048, Abcam) wereused. The same primers were added 5' and 3' to sample DNA of differentlengths through multiple annealing and looping-based amplification cycles(MALBAC) with a 27-nucleotide sequence followed by 8 variablenucleotides (N) as a primer (5′-GTGAGTGATGGTTGAGGTAGTGTG-GAGNNNNNNNN-3′) (Zong et al., 2012), and then quantified by qPCRusing the 27-nucleotide sequence (5′-GTGAGTGATGGTTGAGGTAGT-GTGGAG-3′) as the primer. The DNA precipitated by the anti-lamin B1antibody was quantified as a relative value to the IgG precipitation (the levelof the IgG chromatin immunoprecipitation was set at 1).

Statistical analysisStudent’s t-tests and one-way ANOVA analysis with post hoc Tukeyalgorithm were performed based on the hypothesis of normal distributionand the variances within or between the groups. All data were collected andanalyzed without any preference.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsL.H., K.L., X.J. and L.L. designed the experiments; K.L. and S.M. performed theinitial Drosophila study; L.H. performed the Drosophila and mammalian cell study;Y.L. generated the Drosophila transgenes; and L.H., K.L., X.J. and L.L. wrote themanuscript.

FundingThis work is supported by grants provided to L.L. by the Ministry of Science andTechnology of the People’s Republic of China [grant number 2013CB530700]; andthe National Natural Science Foundation of China for Distinguished Young Scholars[grant number 81325007 to X.J.]

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.184374.supplemental

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