1. Necroptosis
NOH HYUN JIN

2. Cell Death Apoptosis Normal Cell
Autophagy Necrosis
Normal Cell

3. Cell Death

4. As early as the mid-19th century, Rudolf Virchow taught that necrosis is a recognizable form of cell death; since then, pathologists have identified necrosis as both a cause and a consequence of disease. A century later, another form of cell death, apoptosis, was defined, and we now understand that this process is driven by a set of molecular mechanisms that “programs” the cell to die. It has often been assumed that necrosis is distinct from apoptosis, in part because of the belief that necrosis is not programmed by molecular events. It is now clear, however, that in some contexts, necrotic cell death can be driven by defined molecular pathways.
N ENGL J MED 370;

5. Evolution of the Concept of Programmed Necrosis
In some cell types, FAS can trigger non-apoptotic cell death that is independent of caspases but dependent on the adaptor protein FADD and the presence and enzymatic activity of the protein kinase RIP1. This milestone paper is the first report of RIP1‑dependent necroptosis. Nature Immunol, 200, 1: , Tschopp’s group

6. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nature Chem. Biol. 1, 112–119
-- Degterev et al. identify Necrostatin-1 (Nec-1) as a potent inhibitor of TNF mediated necrosis and introduce the term “necroptosis” .

7. Identification of RIP3 as a modulator of Necroptosis
In contrast to RIP1, the loss of murine RIP3 results in no significant phenotypic defect, and has allowed the role of RIP3, and potentially necroptosis, to be examined in vivo during pathogenic infection and inflammatory disease models

8. Identification of MLKL as a modulator of Necroptosis
Identification of MLKL
as a key modulator of necroptosis
NSA
NSA: necrosulfonamide

9. Molecular pathways for Necroptosis

10. TNFR1-mediated necroptosis : the prototype of regulated necrosis

11. Relationship between TRAF2 and TRADD
- Cell line : HeLa(RIP3)
- DNA plasmid: pEGF-c1, GFP-TRAF2, GFP-Peliino (each 1ug)
- Transfection time: 24h, 48h
- Transfection reagent: PEI
GFP
pEGF-c1
GFP-TRAF2
GFP-Pellino
PEI (24h)
PEI (48h)
Actin
TRADD

12. Relationship between TRAF2 and TRADD
- Cell line : HeLa(RIP3)
- DNA plasmid: pEGF-c1, GFP-TRAF2, GFP-Peliino (each 1ug)
- Transfection time: 24h, 36h, 48h
- Transfection reagent: PEI
pEGF-c1
GFP-TRAF2
GFP-Pellino
PEI (24h)
PEI (48h)
PEI (36h)
HeLa (RIP3)
GFP
TRADD (short)
TRADD (long)
Actin (short)
Actin (long)

13. What is RIP3-mediated MLKL activator
under the ER stress condition induced by thapsigargin?

14. What is RIP3-mediated MLKL activator
under the ER stress condition induced by thapsigargin?

15. What is RIP3-mediated MLKL activator
under the ER stress condition induced by thapsigargin?
- Thapsigargin (TG) condition : 9h / 1, 2 (uM) 1 2 - TG
HeLa (RIP3)
pMLKL
RIP3
MLKL
Actin
RIP3
Actin
CHOP 1 2 - TG
HeLa (RIP3)

16. What is RIP3-mediated MLKL activator
under the ER stress condition induced by thapsigargin?
- Thapsigargin (TG) condition : 2uM / 0, 3h, 6h, 9h, 12h - 3 6 9 12 TG
HeLa (RIP3)
(h)
HeLa (NC)
pMLKL (long)
CHOP
MLKL
pMLKL (short)
Actin

17. What is RIP3-mediated MLKL activator
under the ER stress condition induced by thapsigargin?
- Thapsigargin (TG) condition : 12h / 0, 0.5, 1, 2, 4 (uM )
pMLKL (short)
pMLKL (long)
Actin (short)
Actin (long)
CHOP
MLKL -
0.5 1 2 4 TG
HeLa (RIP3)
(uM)

18. What is RIP3-mediated MLKL activator
under the glucose deprivation condition? 12 24 - GD
HeLa (NC)
HeLa (RIP3)
pMLKL (short)
pMLKL (long)
Actin
RIP3
RIP1 (short)
RIP1 (long)
pMLKL
Actin
RIP3
RIP1 - 12 24 GD
HeLa (RIP3)
HeLa (NC)