Skip to main content

Advertisement

SpringerLink
Log in

Single cell analysis of PANoptosome cell death complexes through an expansion microscopy method

  • Original Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatory cell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Data availability

All datasets generated during this study are contained within the figures and supplement of this manuscript.

References

  1. Gullett JM, Tweedell RE, Kanneganti TD (2022) It's All in the PAN: crosstalk, plasticity, redundancies, switches, and interconnectedness encompassed by panoptosis underlying the totality of cell death-associated biological effects. Cells 11(9):1495

  2. Kuriakose T, Man SM, Malireddi RK, Karki R, Kesavardhana S, Place DE, et al (2016) ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways. Sci Immunol 1(2):aag2045

  3. Malireddi RKS, Karki R, Sundaram B, Kancharana B, Lee S, Samir P et al (2021) Inflammatory cell death, PANoptosis, mediated by cytokines in diverse cancer lineages inhibits tumor growth. Immunohorizons 5(7):568–580

    Article  PubMed  Google Scholar 

  4. Kesavardhana S, Malireddi RKS, Burton AR, Porter SN, Vogel P, Pruett-Miller SM et al (2020) The Zα2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development. J Biol Chem 295(24):8325–8330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Banoth B, Tuladhar S, Karki R, Sharma BR, Briard B, Kesavardhana S, et al (2020) ZBP1 promotes fungi-induced inflammasome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis). J Biol Chem 295(52):18276–18283

  6. Christgen S, Zhen, M, Kesavardhana S, Karki R, Malireddi RKS, Banoth B, Place DE, Briard B, Sharma BR, Tuladhar S, Samir P, Burton A, Kanneganti T-D (2020) Identification of the PANoptosome: A molecular platform triggering pyroptosis, apoptosis, and necroptosis (PANoptosis). Front Cell Infect Microbiol 10:237

  7. Karki R, Sharma BR, Lee E, Banoth B, Malireddi RKS, Samir P, et al (2020) Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer. JCI insight 5(12):e136720

  8. Zheng M, Williams EP, Malireddi RKS, Karki R, Banoth B, Burton A, et al (2020) Impaired NLRP3 inflammasome activation/pyroptosis leads to robust inflammatory cell death via caspase-8/RIPK3 during coronavirus infection. J Biol Chem 295(41):14040–14052

  9. Malireddi RK, Ippagunta S, Lamkanfi M, Kanneganti TD (2010) Cutting edge: proteolytic inactivation of poly(ADP-ribose) polymerase 1 by the Nlrp3 and Nlrc4 inflammasomes. J Immunol 185(6):3127–3130

    Article  CAS  PubMed  Google Scholar 

  10. Malireddi RKS, Gurung P, Kesavardhana S, Samir P, Burton A, Mummareddy H, et al (2020) Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity–independent pyroptosis, apoptosis, necroptosis, and inflammatory disease. J Exp Med 217(3):jem.20191644

  11. Malireddi RKS, Kesavardhana S, Karki R, Kancharana B, Burton AR, Kanneganti TD (2020) RIPK1 distinctly regulates yersinia-induced inflammatory cell death, PANoptosis. Immunohorizons 4(12):789–796

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zheng M, Karki R, Vogel P, Kanneganti TD (2020) Caspase-6 is a key regulator of innate immunity, inflammasome activation, and host defense. Cell 181(3):674–87.e13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Karki R, Sharma BR, Tuladhar S, Williams EP, Zalduondo L, Samir P et al (2021) Synergism of TNF-α and IFN-γ triggers inflammatory cell death, tissue damage, and mortality in SARS-CoV-2 infection and cytokine shock syndromes. Cell 184(1):149–68.e17

    Article  CAS  PubMed  Google Scholar 

  14. Lee S, Karki R, Wang Y, Nguyen LN, Kalathur RC, Kanneganti TD (2021) AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence. Nature 597(7876):415–419

  15. Karki R, Sundaram B, Sharma BR, Lee S, Malireddi RKS, Nguyen LN et al (2021) ADAR1 restricts ZBP1-mediated immune response and PANoptosis to promote tumorigenesis. Cell Rep 37(3):109858

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Karki R, Lee S, Mall R, Pandian N, Wang Y, Sharma BR, et al (2022) ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection. Sci Immunol 7(74):eabo6294

  17. Kesavardhana S, Kuriakose T, Guy CS, Samir P, Malireddi RKS, Mishra A et al (2017) ZBP1/DAI ubiquitination and sensing of influenza vRNPs activate programmed cell death. J Exp Med 214(8):2217–2229

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Cookson BT, Brennan MA (2001) Pro-inflammatory programmed cell death. Trends Microbiol 9(3):113–114

    Article  CAS  PubMed  Google Scholar 

  19. Martinon F, Burns K, Tschopp J (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell 10(2):417–426

    Article  CAS  PubMed  Google Scholar 

  20. Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26(4):239–257

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X (1997) Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90(3):405–413

    Article  CAS  PubMed  Google Scholar 

  22. Kim HE, Du F, Fang M, Wang X (2005) Formation of apoptosome is initiated by cytochrome c-induced dATP hydrolysis and subsequent nucleotide exchange on Apaf-1. Proc Natl Acad Sci U S A 102(49):17545–17550

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES et al (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91(4):479–489

    Article  CAS  PubMed  Google Scholar 

  24. Boldin MP, Goncharov TM, Goltsev YV, Wallach D (1996) Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85(6):803–815

    Article  CAS  PubMed  Google Scholar 

  25. Muzio M, Chinnaiyan AM, Kischkel FC, O’Rourke K, Shevchenko A, Ni J et al (1996) FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death–inducing signaling complex. Cell 85(6):817–827

    Article  CAS  PubMed  Google Scholar 

  26. Li H, Zhu H, Xu CJ, Yuan J (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94(4):491–501

    Article  CAS  PubMed  Google Scholar 

  27. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X (1998) Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94(4):481–490

    Article  CAS  PubMed  Google Scholar 

  28. Gross A, Yin XM, Wang K, Wei MC, Jockel J, Milliman C et al (1999) Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J Biol Chem 274(2):1156–1163

    Article  CAS  PubMed  Google Scholar 

  29. Dhuriya YK, Sharma D (2018) Necroptosis: a regulated inflammatory mode of cell death. J Neuroinflamm 15(1):199

    Article  Google Scholar 

  30. Murphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S et al (2013) The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39(3):443–453

    Article  CAS  PubMed  Google Scholar 

  31. Gong Y, Fan Z, Luo G, Yang C, Huang Q, Fan K et al (2019) The role of necroptosis in cancer biology and therapy. Mol Cancer 18(1):100

    Article  PubMed  PubMed Central  Google Scholar 

  32. Nailwal H, Chan FK (2019) Necroptosis in anti-viral inflammation. Cell Death Differ 26(1):4–13

    Article  CAS  PubMed  Google Scholar 

  33. Zhao J, Jitkaew S, Cai Z, Choksi S, Li Q, Luo J et al (2012) Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci USA 109(14):5322–5327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sun L, Wang H, Wang Z, He S, Chen S, Liao D et al (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148(1–2):213–227

    Article  CAS  PubMed  Google Scholar 

  35. Newton K, Wickliffe KE, Dugger DL, Maltzman A, Roose-Girma M, Dohse M et al (2019) Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis. Nature 574(7778):428–431

    Article  CAS  PubMed  Google Scholar 

  36. Man SM, Hopkins LJ, Nugent E, Cox S, Glück IM, Tourlomousis P et al (2014) Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex. Proc Natl Acad Sci USA 111(20):7403–7408

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chen X, Zhu R, Zhong J, Ying Y, Wang W, Cao Y et al (2022) Mosaic composition of RIP1-RIP3 signalling hub and its role in regulating cell death. Nat Cell Biol 24(4):471–482

    Article  CAS  PubMed  Google Scholar 

  38. Man SM, Tourlomousis P, Hopkins L, Monie TP, Fitzgerald KA, Bryant CE (2013) Salmonella infection induces recruitment of Caspase-8 to the inflammasome to modulate IL-1beta production. J Immunol 191(10):5239–5246

    Article  CAS  PubMed  Google Scholar 

  39. Sagulenko V, Thygesen SJ, Sester DP, Idris A, Cridland JA, Vajjhala PR et al (2013) AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC. Cell Death Differ 20(9):1149–1160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Pierini R, Juruj C, Perret M, Jones CL, Mangeot P, Weiss DS et al (2012) AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages. Cell Death Differ 19(10):1709–1721

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wang Y, Kanneganti TD (2021) From pyroptosis, apoptosis and necroptosis to PANoptosis: a mechanistic compendium of programmed cell death pathways. Comput Struct Biotechnol J 19:4641–4657

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Oberst A, Dillon CP, Weinlich R, McCormick LL, Fitzgerald P, Pop C et al (2011) Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature 471(7338):363–367

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Newton K, Sun X, Dixit VM (2004) Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4. Mol Cell Biol 24(4):1464–1469

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ozören N, Masumoto J, Franchi L, Kanneganti TD, Body-Malapel M, Ertürk I et al (2006) Distinct roles of TLR2 and the adaptor ASC in IL-1beta/IL-18 secretion in response to Listeria monocytogenes. J Immunol 176(7):4337–4342

    Article  PubMed  Google Scholar 

  45. Ishii KJ, Kawagoe T, Koyama S, Matsui K, Kumar H, Kawai T et al (2008) TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines. Nature 451(7179):725–729

    Article  CAS  PubMed  Google Scholar 

  46. Jones JW, Kayagaki N, Broz P, Henry T, Newton K, O’Rourke K et al (2010) Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis. Proc Natl Acad Sci USA 107(21):9771–9776

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kanneganti TD, Ozoren N, Body-Malapel M, Amer A, Park JH, Franchi L et al (2006) Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature 440(7081):233–236

    Article  CAS  PubMed  Google Scholar 

  48. Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG (2000) A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci USA 97(11):6108–6113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wang Y, Karki R, Zheng M, Kancharana B, Lee S, Kesavardhana S et al (2021) Cutting edge: caspase-8 is a linchpin in caspase-3 and gasdermin D activation to control cell death, cytokine release, and host defense during influenza A virus infection. J Immunol 207(10):2411–2416

    Article  CAS  PubMed  Google Scholar 

  50. Zhang C, Kang JS, Asano SM, Gao R, Boyden ES (2020) Expansion microscopy for beginners: visualizing microtubules in expanded cultured HeLa cells. Curr Protoc Neurosci 92(1):e96

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Malireddi RKS, Gurung P, Mavuluri J, Dasari TK, Klco JM, Chi H et al (2018) TAK1 restricts spontaneous NLRP3 activation and cell death to control myeloid proliferation. J Exp Med 215(4):1023–1034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Messaoud-Nacer Y, Culerier E, Rose S, Maillet I, Rouxel N, Briault S et al (2022) STING agonist diABZI induces PANoptosis and DNA mediated acute respiratory distress syndrome (ARDS). Cell Death Dis 13(3):269

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Cui Y, Wang X, Lin F, Li W, Zhao Y, Zhu F et al (2022) MiR-29a-3p improves acute lung injury by reducing alveolar epithelial cell PANoptosis. Aging Dis 13(3):899–909

    Article  PubMed  PubMed Central  Google Scholar 

  54. Lin JF, Hu PS, Wang YY, Tan YT, Yu K, Liao K et al (2022) Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis. Signal Transduct Target Ther 7(1):54

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Xu X, Lan X, Fu S, Zhang Q, Gui F, Jin Q et al (2022) Dickkopf-1 exerts protective effects by inhibiting PANoptosis and retinal neovascularization in diabetic retinopathy. Biochem Biophys Res Commun 617(Pt 2):69–76

    Article  CAS  PubMed  Google Scholar 

  56. Chi D, Lin X, Meng Q, Tan J, Gong Q, Tong Z (2021) Real-time induction of macrophage apoptosis, pyroptosis, and necroptosis by Enterococcus faecalis OG1RF and two root canal isolated strains. Front Cell Infect Microbiol 11:720147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Song M, Xia W, Tao Z, Zhu B, Zhang W, Liu C et al (2021) Self-assembled polymeric nanocarrier-mediated co-delivery of metformin and doxorubicin for melanoma therapy. Drug Deliv 28(1):594–606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Masumoto J, Taniguchi S, Ayukawa K, Sarvotham H, Kishino T, Niikawa N et al (1999) ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J Biol Chem 274(48):33835–33838

    Article  CAS  PubMed  Google Scholar 

  59. Chen F, Tillberg PW, Boyden ES (2015) Optical imaging. Expansion microscopy. Science 347(6221):543–548

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Glück IM, Mathias GP, Strauss S, Ebert TS, Stafford C, Agam G, et al (2022) Nanoscale organization of the endogenous ASC speck. bioRxiv. https://doi.org/10.1101/2021.09.17.460822

  61. Samson AL, Fitzgibbon C, Patel KM, Hildebrand JM, Whitehead LW, Rimes JS, et al (2021) A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells. Cell Death Differ 28(7):2126–2144

  62. Bao G, Tang M, Zhao J, Zhu X (2021) Nanobody: a promising toolkit for molecular imaging and disease therapy. EJNMMI Res 11(1):6

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank all members of the Kanneganti laboratory for discussions. We also thank R. Tweedell, PhD, and J. Gullett, PhD, for scientific editing and writing support.

Funding

Research in the Kanneganti laboratory was supported by grants from the US National Institutes of Health (AI101935, AI124346, AI160179, AR056296, and CA253095) and the American Lebanese Syrian Associated Charities to T.D.-K. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Authors and Affiliations

  1. Department of Immunology, St. Jude Children’s Research Hospital, MS #351, 262 Danny Thomas Pl., Memphis, TN, 38105-2794, USA

    Yaqiu Wang, Nagakannan Pandian, Joo-Hui Han, Balamurugan Sundaram, SangJoon Lee, Rajendra Karki, Clifford S. Guy & Thirumala-Devi Kanneganti

Authors
  1. Yaqiu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nagakannan Pandian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joo-Hui Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Balamurugan Sundaram
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. SangJoon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rajendra Karki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Clifford S. Guy
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Thirumala-Devi Kanneganti
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thirumala-Devi Kanneganti.

Ethics declarations

Conflict of interest

T.-D.K. is a consultant for Pfizer.

Ethics approval

Studies were conducted under protocols approved by the St. Jude Children’s Research Hospital committee on the Use and Care of Animals.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

18_2022_4564_MOESM1_ESM.pdf

Supplemental Figure 1. ASC, RIPK3, and CASP8 form a complex under PANoptotic conditions. A) Quantification of the percentage of cells containing ASC specks in IAV-infected wild type (WT) bone marrow-derived macrophages (BMDMs) at 9 h and 12 h post-infection (h.p.i.). B) BMDMs from the indicated genotypes were mock treated, infected with HSV-1, or treated with a KPT-330 + IFN-β and stained for ASC, RIPK3, and CASP8. Representative images of ASC, RIPK3, CASP8-containing specks are shown. C) Quantification of the percentage of cells containing ASC specks in WT BMDMs treated with KPT-330 + IFN-β for 24 h. D) Compositional analysis of ASC specks in (C). Mean ± standard error is shown. Images are representative of at least three independent experiments

18_2022_4564_MOESM2_ESM.pdf

Supplemental Figure 2. “Firework”-like RIPK3 structure forms during LPS + ATP stimulation, and ASC and CASP8 form a core surrounded by RIPK3 during IAV infection. A) Wild type (WT) bone marrow-derived macrophages (BMDMs) were treated with LPS + ATP and stained for ASC, RIPK3, and CASP8. The cells were expanded as described in the methods section. An ASC, RIPK3, and CASP8-containing speck with “firework”-like RIPK3 structure is shown. B) Fluorescence intensity of ASC, RIPK3, and CASP8 across the “firework” section along the white arrow. X axis indicates the distance along the white arrow traveling from the base of the arrow to the arrowhead. C) Quantification of the percentage of cells containing ASC specks in WT BMDMs treated with LPS + ATP for 10 min. D) Compositional analysis of ASC specks (n ≥ 95) induced by LPS + ATP in (C). Mean ± standard error is shown. E) Expansion microscopy views of a RIPK3/CASP8-containing ASC speck induced by IAV. Images are representative of at least three independent experiments

18_2022_4564_MOESM3_ESM.pdf

Supplemental Figure 3. RIPK3 has no observable functional role in LPS + ATP-mediated inflammasome activation. Bone marrow-derived macrophages (BMDMs) from indicated genotypes were primed with 100 ng/μl LPS for 4 h, then stimulated with the indicated concentration of ATP for 30 min. A) Western blots of RIPK3 phosphorylation (Thr231/Ser232; pRIPK3) and total RIPK3 (tRIPK3) in whole cell lysates from indicated genotypes. B) Cell death measured by Sytox Green staining in the indicated genotypes. Microscopy images (right) showing dead cells denoted by red masking. Sytox green detects lytic types of cell death, where the cellular membranes are compromised. C) Caspases-1, -3, -7, -8, GSDMD, and GSDME cleavage, and total and phosphorylated MLKL in whole cell lysates from the indicated genotypes in mock-treated or LPS + ATP-treated cells. Asterisk denotes nonspecific band. D) Quantification of IL-1β and IL-18 release in the supernatant of cells of the indicated genotypes after LPS + ATP treatment. Lysate and supernatant were prepared at 30 min after ATP stimulation. Mean ± standard error is shown. Results are representative of three independent experiments

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, Y., Pandian, N., Han, JH. et al. Single cell analysis of PANoptosome cell death complexes through an expansion microscopy method. Cell. Mol. Life Sci. 79, 531 (2022). https://doi.org/10.1007/s00018-022-04564-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00018-022-04564-z

Keywords

Search

Navigation