Necrotic Pyknosis Is a Morphologically and Biochemically Distinct Event

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SHORT REPORT Necrotic pyknosis is a morphologically and biochemically distinct event from apoptotic pyknosis Lin Hou1,2, Kai Liu1, Yuhong Li2, Shuang Ma2, Xunming Ji2,* and Lei Liu2,*

ABSTRACT triggered by heterogeneous insults, and intermediate morphology Classification of apoptosis and necrosis by morphological differences shared by both apoptosis and necrosis might exist (Galluzzi et al., has been widely used for decades. However, this usefulness of this 2015, 2012; Raffray and Cohen, 1997). method has been seriously questioned in recent years, mainly due Pyknosis has been considered as an irreversible condensation to a lack of functional and biochemical evidence to interpret the of chromatin and the nucleus. It commonly occurs in both morphology changes. To address this matter, we devised genetic apoptotic and necrotic cell death. For apoptosis, the nucleus manipulations in Drosophila to study pyknosis, a process of nuclear usually undergoes condensation, chromatin marginalization and shrinkage and chromatin condensation that occurs in apoptosis and fragmentation into a few large and regular chromatin clumps, which necrosis. By following the progression of necrotic pyknosis, we are eventually packed into apoptotic bodies (Kerr et al., 1972; surprisingly observed a transient state of chromatin detachment from Niquet et al., 2003). In contrast, nuclei in necrotic cells condense the nuclear envelope, followed by the nuclear envelope completely into to smaller chromatin clumps with irregular and dispersed collapsing onto chromatin. This phenomenon led us to discover that morphologies, which might be dissolved later (Bortul et al., 2001; phosphorylation of barrier-to-autointegration factor (BAF) mediates Fujikawa et al., 2000; Hardingham et al., 2002; Niquet et al., 2003). this initial separation of nuclear envelope from chromatin. Therefore, based on the morphology of nuclear fragmentation, Functionally, inhibition of BAF phosphorylation suppressed necrosis pyknosis can be divided into nucleolytic pyknosis (mainly in both Drosophila and human cells, suggesting that necrotic occurring in apoptosis) and anucleolytic pyknosis (mainly pyknosis is conserved in the propagation of necrosis. In contrast, occurring in necrosis) (Burgoyne, 1999). Although pyknosis has during apoptotic pyknosis the chromatin did not detach from the been widely considered as a marker of cell death in vitro and in vivo nuclear envelope and inhibition of BAF phosphorylation had no effect (Colbourne et al., 1999; Fujikawa et al., 1999, 2010; Ji et al., 2013; on apoptotic pyknosis and apoptosis. Our research provides the Niquet et al., 2003; Sohn et al., 1998), it is unclear whether pyknosis first genetic evidence supporting a morphological classification of is a regulated process. apoptosis and necrosis through different forms of pyknosis. Here, we studied the morphological changes visible in pyknosis during the execution of apoptosis and necrosis in a temporal KEY WORDS: BAF phosphorylation, Drosophila genetics, manner. We found that necrotic pyknosis occurred in a distinct Apoptosis, Cell death morphology, Necrosis, Pyknosis pattern compared to apoptotic pyknosis, and phosphorylation of BAF plays a key role only in necrotic pyknosis. INTRODUCTION Morphological differences observed during cell death have been RESULTS AND DISCUSSION widely used to classify apoptosis and necrosis for a long time. These Necrotic pyknosis shows a different morphology to include apoptotic features such as cell shrinkage, membrane apoptotic pyknosis blebbing, nuclear condensation and apoptotic body formation To study necrotic pyknosis, we used a previously established (Kerr et al., 1972), and necrotic features such as cell swelling, necrosis model in Drosophila. By allowing transient expression of plasma membrane rupture, intracellular vacuolization and nuclear a leaky cation channel, the glutamate receptor 1 Lurcher mutant chromatin clumping (Raffray and Cohen, 1997). However, many (GluR1Lc), Ca2+ is overloaded into cells, which results in necrosis exceptions have been discovered and classification of cell death by and fly lethality (Liu et al., 2014). This fly model contains the Gal4 morphology has been controversial (Raffray and Cohen, 1997). driver Appl-Gal4 (expressed in neurons and a few epithelial cells in Recently, the Nomenclature Committee on Cell Death (NCCD) has the larval anal pad), and the UAS expression constructs UAS- recommended using biochemical markers for cell death GluR1Lc and tub-Gal80ts, and we denote it the AG model classification instead of morphology (Galluzzi et al., 2015, 2012). (Appl>GluR1Lc, tub-Gal80ts) (Liu et al., 2014). The AG flies are The reasons include that morphology might not be linked to a healthy at 18°C due to inhibition of Gal4 function by Gal80ts. Upon functional aspect of cell death, a given morphology might be shifting flies to 30°C, Gal80ts is deactivated and necrosis is initiated by GluR1Lc expression (Liu et al., 2014). To label the nuclear

1State Key Laboratory of Membrane Biology, School of Life Sciences, Peking envelope and the chromatin, the fluorescent reporters UAS-koi.GFP University, Beijing 100871, China. 2Aging and Disease Laboratory of Xuanwu and His2Av-mRFP1 (expressing His2Av–mRFP fusion protein in Hospital and Center of Stroke, Beijing Institute for Brain Disorders, Capital Medical all cells) were used, respectively. University, Youanmen, Beijing 100069, China. After being at 30°C for 22 h, the nuclei of control flies [Appl- ts *Authors for correspondence ( [email protected]; [email protected]) Gal4;tub-Gal80 , labeled as wild type (WT); Fig. 1Aa] showed normal morphology, with chromatin occupying the whole nucleus, K.L., 0000-0002-0137-7657; K.L., 0000-0002-6387-7745; X.J., 0000-0002- 0527-2852; L.L., 0000-0002-6387-7745 and the interaction of chromatin with the nuclear envelope was clearly visible. However, after 18 to 20 h at 30°C, the chromatin in

Received 14 December 2015; Accepted 23 June 2016 the epithelial cells of AG flies was dramatically condensed, whereas Journal of Cell Science

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Fig. 1. Characterization of pyknosis in Drosophila. (A) Observation of necrotic pyknosis in vivo in larval anal pad epithelial cells. The nuclear envelope and ts chromatin were labeled by UAS-koi.GFP and H2Av-mRFP1, respectively. (a) The nuclear envelope and chromatin in wild-type (WT) cells (Appl-Gal4;tub-Gal80 ) ts Lc kept at 30°C for 22 h. (b,c) The early stage of nuclear envelope and chromatin changes in AG flies (Appl-Gal4;tub-Gal80 , UAS-GluR1 /cyo) kept at 30°C for the indicated 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 the indicated time. The nuclear envelope and chromatin were labeled by UAS-koi.GFP and H2Av-mRFP1, respectively. (C) BAF protein localization in vivo. The micrographs show the pattern of GFP-tagged wild-type BAF (GFP–BAF-WT) under wild-type (a), AG (b) and AR (c) backgrounds in larval anal pad epithelial cells kept at 30°C for 18 h. In addition, the patterns of GFP–BAF-3A (d) and GFP–BAF-3D (e) under the AG background are shown. (D) List of potential phosphorylation sites 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 experiment shown in E. The bar graph shows the percentage of normal nuclei without nuclear envelope and chromatin disassociation. n=5 experiments with data quantified from four (WT), seven (w1118) and seven (BAF-3A) larvae in each experiment. (G) Effect of BAF-3A on late-stage of necrotic and apoptotic pyknosis in larval anal pad epithelial cells in the indicated lines and conditions. Chromatin changes were revealed by H2Av–mRFP1. White dotted lines mark the boundary of the anal pad. (H) Quantification of data from the experiment shown in G. The bar graph shows the number of fragmented, anucleolytic pyknotic 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<0.01; ***P<0.001; NS, not significant (two-tailed one-way ANOVA with post hoc Tukey algorithm was performed for comparison between three or more

samples; for two sample comparisons, a two-tailed Student’s t-test was used). Journal of Cell Science

3085 SHORT REPORT Journal of Cell Science (2016) 129, 3084-3090 doi:10.1242/jcs.184374 the nuclear envelope only slightly shrank, leading to the detachment morphology defect of necrotic pyknosis was completely abolished of chromatin from the nuclear envelope (Fig. 1Ab,c). Later, both the by the BAF-3A expression (Fig. 1Ec,F). Similarly, expression of nuclear envelope and the chromatin were further compacted BAF-3A rescued the late-stage morphology of necrotic pyknosis and eventually collapsed together (Fig. 1Ad). The morphology is (Fig. 1Ga–c,H). For apoptotic pyknosis, BAF-3A expression had no consistent with the features of anucleolytic pyknosis (Fujikawa inhibitory effect (Fig. 1Gd,e,H), indicating different molecule(s) et al., 2010; Sohn et al., 1998). might be required. We also studied apoptotic pyknosis in vivo by transient expression of reaper (rpr)(Appl>rpr, tub-Gal80ts, denoted AR), Anucleolytic pyknosis plays a functional role in necrotic cell which induces classical apoptosis (White et al., 1996). We found death that the nuclear envelope and chromatin physically shrank together, Although anucleolytic pyknosis has been considered to be a marker with the nucleus eventually fragmenting into regular clumps of necrosis, its function in cell death is unclear. To address this (Fig. 1Ba–d). question, we investigated the survival of AG flies under different transgenic BAF mutant backgrounds. Transient overexpression of Phosphorylation of BAF specifically mediates necrotic BAF-3A or BAF-3D in neurons had no effect on fly survival pyknosis (Fig. 2A). Upon induction of necrosis, the survival rate of adult AG Our data suggest that necrotic pyknosis is likely to be initiated by the flies decreased to 38.4% (Fig. 2A). Overexpression of BAF-WT detachment of chromatin from the nuclear envelope. Previous resulted in a similar survival rate (Fig. 2A). Strikingly, the studies have shown that barrier-to-autointegration factor (BAF) overexpression of BAF-3A increased the survival rate to 70.0%, plays a key role in chromatin tethering to the nuclear envelope whereas the expression of BAF-3D reduced the survival rate to through its interaction with LAP2, emerin and MAN1 (LEM)- 11.7% (Fig. 2A). At the cellular level, BAF-3A suppressed the domain-containing proteins and double-stranded DNA (dsDNA) in necrotic morphology, whereas BAF-3D enhanced it, and BAF-WT a sequence non-specific manner (Umland et al., 2000; Zheng et al., had no effect (Fig. 2B,C). Taken together, BAF phosphorylation 2000). alone is not sufficient to induce cell death, but it is necessary for Based on the function of BAF, it is possible that the dissociation necrotic pyknosis and the propagation of cell death. In contrast, the of chromatin from the nuclear envelope is induced by deregulation expression of BAF-3A or BAF-3D had no effect on fly models of of BAF during necrosis. To examine BAF localization in necrosis, apoptosis, including the eye defects of GMR-Hid (Grether et al., we generated a GFP-tagged BAF transgene (UAS-GFP-BAF). It has 1995) and GMR>eiger (Hid and Eiger induce caspase-mediated and been reported that an N-terminal GFP tag does not affect BAF JNK-mediated apoptosis, respectively; Moreno et al., 2002) activity (Nichols et al., 2006; Shimi et al., 2004). In wild-type cells (Fig. 2D). (Appl-Gal4;tub-Gal80ts) after 30°C for 18 h, GFP–BAF was Owing to a lack of antibody that can directly detect the distributed on chromatin and interacted with the nuclear envelope phosphorylation of Drosophila BAF, we assessed BAF (Fig. 1Ca). In the AG flies, most GFP–BAF protein was also phosphorylation by immunoprecipitating GFP–BAF proteins with localized on the compacted chromatin (Fig. 1Cb), suggesting that an anti-GFP antibody followed by western blotting with an antibody BAF localization is not drastically altered during necrosis. However, against phosphorylated threonine (pThr). The result showed that GFP–BAF reduced its distribution in the chromatin and formed BAF phosphorylation indeed increased in the AG flies, and the ring-like structures in apoptosis (Fig. 1Cc). This phenomenon is expression of BAF-3A abolished the phosphorylation (Fig. 2E). The consistent with a previous report stating that BAF disassembled and subtle change in BAF phosphorylation is likely due to only ∼1% of disappeared from the nucleus during apoptosis (Furukawa et al., neurons undergoing necrosis, which is sufficient to cause fly 2007). lethality in the AG flies (Liu et al., 2014). No signal could be The N-terminal phosphorylation of BAF has been reported to detected when an antibody against phosphorylated serine was cause the detachment of chromatin from the nuclear envelope applied. This result suggests that threonine 4 in the N-terminal of during karyosome formation in the meiosis of the oocyte in BAF is the functional site that is phosphorylated during necrotic Drosophila (Lancaster et al., 2007). The BAF N-terminus has three pyknosis. To test this hypothesis, we generated transgenes to express potential phosphorylation sites, including serine 2, threonine 4 and BAF proteins with both threonine 4 and serine 5 mutated to alanine serine 5. To test the importance of BAF phosphorylation in necrotic (BAF-2A) or threonine 4 alone mutated to alanine (BAF-1A) pyknosis, we generated transgenic flies expressing BAF proteins (Fig. 1D). We found that both BAF-2A and BAF-1A could rescue with these three potential phosphorylation sites mutated to alanine the lethality of the AG flies, similar to BAF-3A (Fig. 2A). (the non-phosphorylatable form, denoted BAF-3A) or aspartic acid (the phospho-mimic form, denoted BAF-3D). These mutation sites BAF phosphorylation regulates necrotic pyknosis in of BAF are illustrated in Fig. 1D. When these mutant flies were mammalian cells crossed to AG flies to induce necrosis, GFP–BAF-3A localization Because of the conserved function of BAF in chromatin anchoring appeared to be similar to the pattern of wild-type BAF (GFP–BAF- in metazoans (Segura-Totten and Wilson, 2004), we asked whether WT) under normal conditions (Fig. 1Cd), whereas GFP–BAF-3D BAF phosphorylation regulates necrotic pyknosis in mammalian showed a similar dissociation phenotype to that of the GFP–BAF- cells. To induce necrosis, human neuroblastoma SH-SY5Y cells WT in the AG background (Fig. 1Ce). These data suggest that were treated with Ca2+ ionophore to overload Ca2+.Ca2+ ionophore chromatin disassociation from nuclear envelope during necrosis is treatment led to nuclear shrinkage into propidium-iodide-positive, likely inhibited by GFP–BAF-3A expression. To further confirm small, round and bright chromatin clumps within 30 min the role of BAF-3A, we quantified the number of cells with early (Fig. 3Aa–b′). However, we did not observe the transient stage necrotic pyknosis features in cells with the chromatin and detachment of chromatin from the nuclear envelope in SH-SY5Y nuclear envelope labeled by His2Av–mRFP1 and koi.GFP. In the cells and in several other cultured mammalian cells (Fig. 3Ac; AG flies, 58% of cells displayed the early morphological features of Fig. S1). This variation might be due to the difference between necrotic pyknosis (Fig. 1Ea,b,F). Strikingly, the early stage in vivo and in vitro systems. In tissues, the cellular contacts play an Journal of Cell Science

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Fig. 2. Functional role of BAF phosphorylation on necrosis and apoptosis. (A) The effect of expression of BAF mutants on the survival of AG flies. The flies were incubated 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- ts Gal80 )andn=7 for the AG flies. Fifty flies were tested for each experiment. (B) The effect of the BAF mutants on necrosis in larval anal pads. Larvae were incubated at 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,andan earlier 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 of cell 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-dependent DN apoptosis and the JNK-dependent cell death models in Drosophila eyes. Overexpression of IAP1 and bsk are shown as the positive controls, which suppress the indicated cell death pathways. (E) Immunoprecipitation (IP) followed by immunoblotting (WB) to detect BAF phosphorylation at threonine residues (pThr) during necrosis. A representative experiment from n=3 is shown. The asterisk indicates a non-specific band present in all samples, which runs slightly higher than the phosphorylated GFP–BAF band (<). Data in all bar graphs are given as the means+s.d. **P<0.01; ***P<0.001; NS, not significant (two-tailed one-way ANOVA with post hoc Tukey algorithm was performed for comparison between three or more samples; for two sample comparisons, a two-tailed Student’s t-test was used). important role in setting the threshold of cell death (Raffray and the nucleus is highly compacted and the dissociation of nuclear Cohen, 1997). In fact, the intermediate state of the nuclear envelope from chromatin might be less obvious at the cell envelope being detached from chromatin has been observed in the morphological level. To study the interaction between the retinoblastoma patients (Buchi et al., 1994). In mammalian cells, nuclear envelope and chromatin in mammalian cells, the DNA Journal of Cell Science

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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′ and b,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) for 30 min. b and b′ show the chromatin condensation (DAPI) in necrotic cells (propidium iodide positive). (c) Co-staining of chromatin (DAPI) and the nuclear envelope (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, as assessed by agarose gel electrophoresis. Results are means±s.d. relative to the level of the control (IgG precipitation without adding Ca2+ ionophore) which was set 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 the chromatin immunoprecipitation (chIP) DNA fraction was normalized to its own input DNA fraction. The DNA level of the normalized background (IgG chIP) was set as 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 mutation sites. (E) Effect of BANF1 on necrosis. Stable human SH-SY5Y cell lines expressing GFP (a,b), wild-type BANF1 (BANF1-WT) (c) or non-phosphorylatable BANF1 (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 lower panels are the same views with DAPI and propidium iodide staining, respectively. (F) Quantification of results shown in E. n=3 experiments; data were quantified from 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<0.05; **P<0.01; ***P<0.001; NS, not significant (two-tailed one-way ANOVA with post hoc Tukey algorithm was performed for comparison between three or more ’ t samples; for two sample comparisons, a two-tailed Student s -test was used). Data shown in panels C, F and G are given as the mean±s.d. Journal of Cell Science

3088 SHORT REPORT Journal of Cell Science (2016) 129, 3084-3090 doi:10.1242/jcs.184374 that tethered on the inner nuclear membrane was precipitated by DFFB), endonuclease G and DNase I in nuclear fragmentation an anti-lamin-B1 antibody. Then, the precipitated DNA was (Enari et al., 1998; Li et al., 2001; Liu et al., 1997; Oliveri et al., quantified by an agarose gel and a quantitative PCR (qPCR) assay. 2001), and phospholipase A2 in necrotic pyknosis (Shinzawa and The result showed that the amount of lamin-B1-bound DNA Tsujimoto, 2003). Here, we provide an example of morphological was significantly decreased during necrosis in the SY5Y cells regulation of necrotic pyknosis by a biochemical event. (Fig. 3B,C). This result indicates that the dissociation of chromatin and nuclear envelope might also take place during necrosis in the MATERIALS AND METHODS mammalian cells. Drosophila stocks and maintenance The potential phosphorylation sites of human BAF (BANF1) Flies were raised on standard cornmeal medium. The stocks were kindly include threonine 2, threonine 3 and serine 4 (Fig. 3D). Because the provided by colleagues including: UAS-eiger (Lei Xue, School of Life site equivalent to the Drosophila BAF threonine 4 was unclear Science and Technology, Tongji University, China), GMR-hid, GMR-Gal4 (Andreas Bergmann, Department of Cancer Biology, University of (Lancaster et al., 2007), we mutated all three sites (Fig. 3D, BANF1- Massachusetts Medical School, Worcester, MA) and UAS-IAP1 (Denise 3A), and expressed the mutant constructs in SH-SY5Y Montell, Molecular, Cellular, and Developmental Biology, University of cells. The result showed that expression of BANF1-3A (a non- California, Santa Barbara, CA). We generated the following lines from the phosphorylatable mutant) abolished the reduction of lamin-B1- w1118 background by P-element insertion: UAS-GFP-BAF-WT, UAS-GFP- bound DNA during necrosis (Fig. 3B,C). To examine the functional BAF-3A, UAS-GFP-BAF-3D, UAS-BAF-WT-Flag, UAS-BAF-3A-Flag, role of BANF1 phosphorylation on necrosis, we performed Ca2+ UAS-BAF-3D-Flag, UAS-GFP-BAF-2A and UAS-GFP-BAF-1A. UAS-koi. ionophore experiments. Ca2+ ionophore treatment induced ∼30.4% GFP (BL#26266) and H2Av-mRFP1 (BL#23650) were obtained from the propidium-iodide-positive nuclei (Fig. 3E,F). However, expression Bloomington Drosophila Stock Center. of BANF1-3A reduced the proportion of cells that died to 14.4% (Fig. 3Ec,d,F). BANF1-3A also blocked the ATP depletion upon Protein extraction and immunoprecipitation All cell lines were obtained from the China Infrastructure of Cell Line Ca2+ ionophore treatment (Fig. 3G). – Resources and tested for contamination. Fly heads and human SH-SY5Y To examine BANF1 phosphorylation, BANF1 Flag proteins were cells treated with Ca2+ ionophore (A23187; Tocris Bioscience #1234) were precipitated with an anti-Flag antibodyand assessed with an anti-pThr lysed in buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5% NP40) antibody. The result showed that the phosphorylation of BANF1- supplemented with protease inhibitor cocktail (Roche) and phosphatase – WT Flag was not detectable under normal conditions but it was inhibitors (1 mM Na3VO4, 50 mM NaF, 30 mM glycerophosphate and greatly increased upon treatment with Ca2+ ionophore (Fig. 3H). 0.5 mM EDTA). After sonication and centrifugation, the supernatant was Importantly, there was no detectable phosphorylation of BANF1 incubated with normal IgG (1:1000, Santa Cruz Biotechnology, sc-2025) when BANF1-3A–Flag was expressed (Fig. 3H). This result indicates and protein-A/G–agarose beads (Thermo Scientific Pierce, #20421). After that BAF phosphorylation is a conserved event of necrosis. centrifugation, the supernatant was incubated with anti-GFP antibody Our study identifies BAF phosphorylation as a specific (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 were biochemical marker for necrotic pyknosis in certain types of cells, added and incubated overnight. After washing with lysis buffer six times, suggesting that classical apoptotic and necrotic pyknosis might be the pellet was boiled in SDS loading buffer. Tricine-SDS-PAGE was used to distinct processes at the molecular level. A schematic model of separate the small-molecular-mass proteins. BAF phosphorylation was necrotic pyknosis is shown in Fig. 4. For classification of cell death, detected with an anti-pThr antibody (1:500, Cell Signaling #9381). identification of more biochemical markers that directly regulate morphology should greatly improve the uncertainty in categorizing Nuclear morphology and cell survival in cultured cells more complicated modes of cell death (Raffray and Cohen, 1997). Antibody against the nuclear pore complex (NPC) (1:1000, Abcam In fact, several regulators of apoptosis and necrosis have been ab24609, clone Mab414) was used for immunostaining. SH-SY5Y cells identified, including caspase-activated DNase (CAD, also known as were 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, BAF phosphorylation promotes condensed chromatin to dissociate from the nuclear envelope. Then, at later stages, the nuclear envelope collapses onto the chromatin and the plasma membrane is damaged. BAF-ph, phosphorylated BAF. Journal of Cell Science

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ATP level was determined by a CellTiter-Glo® Luminescent Cell Viability Fujikawa, D. G., Zhao, S., Ke, X., Shinmei, S. S. and Allen, S. G. (2010). Mild as Assay kit (Promega). The relative ATP level (%)=ATP [with Ca2+ well as severe insults produce necrotic, not apoptotic, cells: evidence from 60-min Neurosci. Lett. ionophore]/ATP [without Ca2+ ionophore]×100. seizures. 469, 333-337. Furukawa, K., Aida, T., Nonaka, Y., Osoda, S., Juarez, C., Horigome, T. and Sugiyama, S. (2007). BAF as a caspase-dependent mediator of nuclear Chromatin immunoprecipitation and quantitative PCR apoptosis in Drosophila. J. Struct. Biol. 160, 125-134. After Ca2+ ionophore or DMSO treatment for 30 min, chromatin Galluzzi, L., Vitale, I., Abrams, J. M., Alnemri, E. S., Baehrecke, E. H., immunoprecipitation was performed as described previously (Boyd et al., Blagosklonny, M. V., Dawson, T. M., Dawson, V. L., El-Deiry, W. S., Fulda, 1998). Protein A+G agarose, salmon sperm DNA (Merck Millipore S. et al. 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The using the 27-nucleotide sequence (5′-GTGAGTGATGGTTGAGGTAGT- head involution defective gene of Drosophila melanogaster functions in Genes Dev. GTGGAG-3′) as the primer. The DNA precipitated by the anti-lamin B1 programmed cell death. 9, 1694-1708. Hardingham, G. E., Fukunaga, Y. and Bading, H. (2002). Extrasynaptic NMDARs antibody was quantified as a relative value to the IgG precipitation (the level oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. of the IgG chromatin immunoprecipitation was set at 1). Nat. Neurosci. 5, 405-414. Ji, W.-T., Lee, C.-I., Chen, J. Y.-F., Cheng, Y.-P., Yang, S.-R., Chen, J.-H. and Statistical analysis Chen, H.-R. (2013). Areca nut extract induces pyknotic necrosis in serum-starved Student’s t-tests and one-way ANOVA analysis with post hoc Tukey oral cells via increasing reactive oxygen species and inhibiting GSK3beta: an implication for cytopathic effects in betel quid chewers. PLoS ONE 8, e63295. algorithm were performed based on the hypothesis of normal distribution Kerr, J. F. R., Wyllie, A. H. and Currie, A. R. (1972). Apoptosis: a basic biological and the variances within or between the groups. All data were collected and phenomenon with wide-ranging implications in tissue kinetics. Br.J. Cancer 26, analyzed without any preference. 239-257. Lancaster, O. M., Cullen, C. F. and Ohkura, H. (2007). NHK-1 phosphorylates BAF Competing interests to allow karyosome formation in the Drosophila oocyte nucleus. J. Cell Biol. 179, The authors declare no competing or financial interests. 817-824. Li, L. Y., Luo, X. and Wang, X. (2001). Endonuclease G is an apoptotic DNase Nature Author contributions when released from mitochondria. 412, 95-99. Liu, X., Zou, H., Slaughter, C. and Wang, X. (1997). DFF, a heterodimeric protein L.H., K.L., X.J. and L.L. designed the experiments; K.L. and S.M. performed the that functions downstream of caspase-3 to trigger DNA fragmentation during initial Drosophila study; L.H. performed the Drosophila and mammalian cell study; apoptosis. Cell 89, 175-184. Y.L. generated the Drosophila transgenes; and L.H., K.L., X.J. and L.L. wrote the Liu, K., Ding, L., Li, Y., Yang, H., Zhao, C., Lei, Y., Han, S., Tao, W., Miao, D., manuscript. Steller, H. et al. (2014). Neuronal necrosis is regulated by a conserved chromatin- modifying cascade. Proc. Natl. Acad. Sci. USA 111, 13960-13965. Funding Moreno, E., Yan, M. and Basler, K. (2002). Evolution of TNF signaling This work is supported by grants provided to L.L. by the Ministry of Science and mechanisms: JNK-dependent apoptosis triggered by eiger, the drosophila Technology of the People’s Republic of China [grant number 2013CB530700]; and homolog of the TNF superfamily. Curr. 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