. 2021 May 28;11(6):803. doi: 10.3390/biom11060803

Table 1.

Understanding the role of necroptosis in the aetiology of disease using Mlkl^−/− and mutant mice.

Challenge	Method	Outcome in Mlkl^−/− (Mutant or Knock-Down) Mouse Relative to Wild-Type Control	Mlkl^−/− Mouse and Wild-Type Control Details	Reference
Ischemia and reperfusion injury (IRI)
Hepatic IRI	45 min ischemia in chow or Western diet fed mice.	Reduced TUNEL positive hepatocytes and serum ALT levels in Mlkl^−/− mice, irrespective of diet. Hepatic neutrophil infiltration and TNF, IL-1, MIP-2, and IL-6 mRNA reduced post I/R in Mlkl^−/− mice.	Sex-matched Mlkl^−/− (CRISPR-Cas9) and Wt mice utilised.	[17]
Renal infarction, no reperfusion	Cholesterol crystal embolism.	Mlkl^−/− mice protected from infarction (measured by infarct size, kidney injury and neutrophil infiltration). No difference in the extent of acute kidney injury (measured by eGFR) and kidney failure.	Mlkl^−/− [5] and Wt mice utilised.	[18]
Renal IRI	40 min bilateral renal pedicle clamp.	Mlkl^−/− mice protected from acute kidney injury (measured using serum creatinine and urea). Ripk3^−/−Mlkl^−/− double knockout mice were only somewhat protected.	Sex- and weight-matched Wt controls for Mlkl^−/− [5] mice.	[19]
Renal IRI	30 min renal pedicle clamp.	Mlkl^−/− prolonged survival by ~4 days over Wt mice.	Male Wt and Mlkl^−/− [5] littermates.	[20]
Focal cortical ischemia	Photothrombosis of the cortical microvessels.	Reduced infarction (fewer TUNEL/NeuN-positive cells) in Mlkl^−/− mice and improved locomotion from day 7. Fewer F4/F80+ cells and reduced expression of iNOS, TNFα, IL-12 and IL-18.	Mlkl^−/− sourced from Xiamen University *	[21]
Sterile Inflammatory
Acute kidney injury (AKI)	Intraperitoneal injection of sodium oxalate.	Mlkl^−/− mice show reduced serum creatinine, neutrophil recruitment, and tubular necrosis (TUNEL staining and tubular injury score).	Sex matched male Wt controls for Mlkl^−/− [5] mice.	[22]
Acute kidney injury (AKI)	Intraperitoneal injection of cisplatin.	Mlkl^−/− mice resistant to tubular necrosis (histology, blood urea nitrogen and serum creatinine). Reduced TNFα, IL-1β, IFN-γ, IL-6 in proximal tubules despite similar levels to Wt at baseline.	Male Wt and Mlkl^−/− (TALEN) mice are littermates.	[23]
Hepatitis	ConA-induced.	Mlkl^−/− mice show reduced hepatocyte necrosis (TUNEL staining and ALT/AST).	Wt and Mlkl^−/− [5] a combination of C57BL/6J or littermate controls used.	[24]
	ConA-induced.	Hepatocyte necrosis in Mlkl^LPC-KO mice, with specifically ablated MLKL in liver parenchymal cells, are indistinguishable from Wt (ALT and H&E).	Mlkl^LPC-KO and Mlkl^fl/fl are littermates.	[25]
	LPS/GalN-driven (model of apoptotic hepatitis).	Mlkl^−/− mice are indistinguishable from Wt (assessed using ALT/AST and histological staining for cleaved Casp3).	Wt and Mlkl^−/− [5] a combination of C57BL/6J or littermate controls used.	[24]
	Acetaminophen (model of drug-induced liver injury).	MLKL deficiency does not prevent liver injury (assessed using AST/ALT levels and TUNEL staining).	Wt and Mlkl^−/− [5] a combination of C57BL/6J or littermate controls used.	[24]
	Acetaminophen (model of drug-induced liver injury).		Male Wt used for Mlkl^−/− [5] mice. Bred as separate cohorts.	[26]
Acute pancreatitis	Cerulein-induced.	Mlkl^−/− mice experience less severe acinar cell necrosis than Wt mice (as measured by histological and quantitative analysis).	Male Mlkl^−/− (TALEN) and Wt mice are littermates.	[7]
ANCA-driven necrotising crescent glomerulonephritis	αMPO IgG transfer.	Mlkl^−/− mice were protected against NCGN (as measured by reduced leukocyturia, erythrocyturia and less crescents and necrotic change on histology).	Mlkl^−/− [5]. *	[27]
Arthritis	K/B X N serum transfer.	No significant difference in disease progression between Mlkl^−/− and Wt mice (clinical severity scale and myeloperoxidase average radiance).	Sex matched Wt for Mlkl^−/− mice.	[28]
Arthritis	Myeloid-cell-specific A20 deficiency (A20^MYC-KO).	A20^MYC-KOMlkl^−/− mice were protected against inflammatory arthritis (thickness of rear ankles, histological scores of inflammation, cartilage and bone destruction), splenomegaly and showed reduced expression of IL-1β and TNFα.	Mice with loxP-flanked A20 crossed with Mlkl^−/− [29] aged to 23-to-27 weeks old.	[30]
Colitis and ileitis	IEC-specific FADD and Caspase-8 deficiency.	Mlkl^−/−Fadd^IEC-KO and Mlkl^−/−Casp8^IEC-KO resistant to colitis. Mlkl^−/−Casp8^IEC-KO are also resistant to ileitis whilst Mlkl^−/−Fadd^IEC-KO are only partially protected. Assessed using histological analysis of colonic and ileal tissues and microarray.	Littermate control mice with homozygous or heterozygous loxP-flanked Fadd or Casp8.	[31]
Colitis and ileitis	IEC-specific highly active mTORC1 pathway (Tsc1^IEC-KO).	Tsc1^IEC-KOMlkl^−/− profoundly alleviates epithelial cell death and intestinal barrier dysfunction that is seen in Tsc1^IEC-KO.	Tsc1^IEC-KO and Tsc1^IEC-KOMlkl^−/− were age- and sex-matched littermate controls.	[32]
Lethal ileitis	X-linked transgene -short form of cellular FLICE-inhibitory protein (cFLIPs).	MLKL deficiency partially rescues male mice from lethal in-utero ileitis (improved survival, small intestine villous architecture, fewer CC3-positive cells).	Male Wt and Mlkl^−/− [33].	[34]
Acute colitis	1.5% DSS in drinking water, 5 days, followed by 3 days of normal drinking water.	Mlkl^−/− mice display increased weight loss, but only in some cohorts. Day 5 and 8, indistinguishable murine endoscopic index of colitis (MEIC) scores. Increased inflammation in the proximal colon and decreased submucosal inflammation in the distal colon (H&E).	Wt and Mlkl^−/− [5] are littermates.	[35]
Inflammatory skin disease	RIPK1 deficient keratinocytes Ripk1^EKO.	Specific MLKL deficiency in keratinocytes profoundly ablated the development of the inflammatory skin lesion that invariably develops in Ripk1^EKO mice.	Ripk1^FL/FL (Takahashi et al., 2014) Mlkl^FL/FL (Murphy et al., 2013) Wt littermates utilised.	[36]
Systemic inflammatory response syndrome (SIRS)	300 μg/kg TNF intravenous/kg TND.	Mlkl^−/− mice suffer a hypothermia reaction like that of Wt controls.	Wt and Mlkl^−/− [5] are littermates.	[20]
	500 μg/kg TNF intravenous.	Mlkl^−/− mouse core body temperature higher at 6, 8 and 10 h post dose than Wt controls, indicating moderate protection.	Wt and Mlkl^−/− [5] are littermates.	[20]
	30 mg/kg LPS-intraperitoneal.	Mlkl^−/− mice show similar serum TNFα and IL-1β levels as Wt (measured at 0, 2, 4 and 8 h by ELISA).	Male Mlkl^−/− (TALEN) and Wt mice are littermates.	[7]
	1 mg/kg TNF in 200 μL PBS via the tail vein.	Mlkl^−/− mice showed increased survival. Ripk3^−/−Mlkl^−/− double knockout mice were not similarly protected.	Female Wt and Mlkl^−/− [5] mice bred as separate cohorts.	[19]
	50 μg/kg LPS intraperitoneal.	Mlkl^−/− mice showed comparable levels of TNFα and IL-6 (ELISA analysis) 1 h post-treatment. Pre-treatment with necrostatin-1 (IV), 15 min prior to LPS, significantly reduced Mlkl^−/− mice ability to produce TNFα and IL-6.	*	[37]
Generalised inflammation	A20 gene deficiency.	Mlkl^−/−A20^−/− mice display similar inflammation (hepatic neutrophil infiltration, RANTES secretion and dermatitis) and lifespan.	Wt are A20^−/− littermates.	[20]
TNF-induced multi-organ inflammation	SHARPIN deficiency shpn^m/m.	12-week-old Shpn^m/mMlkl^−/− mice show reduced liver inflammation, splenomegaly and leucocytosis in comparison to Shp^m/m controls (histology, ADVIA automated hematological analysis).	C57BL/Ka Shpn^cpdm/cpdm backcrossed to C57BL/6J one or two times or crossed with C56BL/6J Mlkl^−/− [5] mice.	[38]
Casp8^D387A/D387A (non-cleavable caspase 8)-induced systemic inflammation	Casp8^D387A/D387A mutation.	Mlkl^−/− Casp8^DA/DA do not develop LPR disease. Mlkl^−/− Casp8^DA/DA show an exacerbated inflammatory phenotype with significant splenomegaly, liver damage (serum ALT, AST) and premature death.	Casp8^D387A/D387A (CRISPR-Cas9) and Mlkl^−/− [5] mice are sex-matched.	[39]
Mlkl^D139V-induced lethal neonatal inflammatory syndrome	Whole body homozygosity for constitutively active Mlkl^D139V mutation.	Mlkl^D139V homozygotes are born normal but develop acute multifocal inflammation of the head, neck, and mediastinum by day P2/P3. Maximum lifespan observed 6 days.	Age and sex matched littermate controls.	[40]
Infection
Bacterial
Staphylococcus aureus	Subcutaneous 2 × 10⁶ CFU of S. aureus strain MRSA USA300.	5-day p.i. Mlkl^−/− mice suffer greater bacterial burdens, larger skin lesions, significantly increased neutrophil, macrophage, γδ T cell infiltrate and enhanced pro-inflammatory cytokine expression.	Sex-matched Wt and Mlkl^−/− [5].	[41]
	Retro-orbital 1 × 10⁸ CFU S. aureus strain MRSA USA300.	Mlkl^−/− mice show increased mortality rates compared to WT (a mean survival of 4 days).	Sex-matched Wt and Mlkl^−/− [5].	[41]
	Intravenous 1 × 10⁷ CFU of S. aureus strain MRSA.	Accelerated weight loss and morbidity in Mlkl^−/− mice. Circulating neutrophil numbers elevated 24 h p.i. Increased CFU in the blood and MRSA burden in the kidney of Mlkl^−/− mice.	Mlkl^−/− [5] mice littermates utilised.	[42]
	Retro-orbital 1 × 10^6–7 CFU of S. aureus strain MRSA.	Mlkl^−/− mice show increased MRSA burden in blood and kidneys and greater number of peripheral neutrophils (flow cytometry).	Mlkl^−/− [5] mice littermates utilised.	[42]
	Intraperitoneal 1 × 10⁷ CFU of S. aureus strain MRSA.	Mlkl^−/− mice display more severe bacteraemia 24 h p.i.	Mlkl^−/− [5] mice littermates utilised.	[42]
Mycobacterium tuberculosis	~100–200 CFU of Mtb via aerosol strain H37Rv.	Mlkl^−/− mice are indistinguishable from Wt controls in terms of splenic and respiratory bacterial burden, gross lung histopathology of inflammatory lesions (number and size), organisation of granulomatous inflammation, immune cell counts, TNFα and IL-1β.	Sex-matched Wt and Mlkl^−/− [5] mice.	[43]
Polymicrobial septic shock	Cecal ligation and puncture (CLP).	Mlkl^−/− and Wt mice showed same survival profile (mortality was monitored from 24 h to 144 h).	Age- and sex-matched littermates of Wt and Mlkl^−/−.	[7]
Polymicrobial septic shock	Cecal ligation and puncture (CLP).	Mlkl^−/− have better survival (~50%) than Wt controls (~25%) at 180 h and are moderately protected against hypothermia. Mlkl^−/− experience less severe lung, small intestine (H&E) injury with reduced levels of ALT, serum BUN, HMGB1, TF, IL-1β, TNFα.	Age- and sex-matched Wt and Mlkl^−/−.	[44]
Acute kidney injury (AKI) following polymicrobial septic shock	Cecal ligation and puncture (CLP).	Mlkl^−/− mice suffer same extent of AKI. Ratio of lipocalin-2/urine creatinine levels were lower in Mlkl^−/− mice than Wt, however not as reduced as Ripk3^−/− mice.	Wt and Mlkl^−/− (Jiahuai Han laboratory) littermates.	[45]
Asymptomatic chronic nasopharyngeal colonisation with S. pneumoniae	Nasal instillation of ~1 × 10⁵ CFU of serotype 4 strain TIGR4 m.	Mlkl^−/− mice demonstrate reduced nasopharyngeal epithelial cell sloughing and increased LDH, Il-33, IL-1α, CXCL2 levels and decreased Il-6, Il-17 and polymorphonuclear cells than Wt. Mlkl^−/− mice cleared S. pneumoniae colonisation at a slower rate than Wt controls. Less anti-PspA IgG than Wt controls despite comparable total serum IgG concentration.	Wt and Mlkl^−/− [5] mice utilised.	[46]
S. marcescens hemorrhagic pneumonia	Intratracheal infection with strain MB383.	Mlkl^−/− mice have increased alveolar macrophages and suffer less lung damage (histological analysis).	Wt and Mlkl^−/− littermates (Douglas Green).	[47]
Listeria monocytogenes	Oral 1 × 10⁸ CFU L. monocytogenes 1/2b strain 2011L-2858.	3 days p.i. Mlkl^−/− mice have a moderate increase in liver bacterial colonisation compared to Wt littermate controls.	Female Wt and Mlkl^−/− [5] littermates.	[48]
Salmonella enterica	Oral 5 × 10⁷ CFU −1 × 10⁸ CFU Subsp. Enterica serovar Typhimurium strain SL1344.	Mlkl^−/− mice show greater submucosal oedema, loss of goblet cells, PMN infiltration, loss of epithelial barrier integrity and salmonella colonisation despite comparable bacterial faecal loads. Also have increased body and cecal weight loss.	Wt and Mlkl^−/− (Dr. Jia-Huai Han, Xiamen University, China) were sex-matched.	[49]
Viral
Influenza A strain PR8	Instranasal 4000 EID50.	Mlkl^−/− mice indistinguishable from Wt in terms of survival (75% of both Mlkl^−/− and Wt mice survived and recovered) and lung progeny virion output.	Sex-matched Wt controls *.	[50]
	Instranasal 2500 EID₅₀. Intranasal 6000 EID₅₀.	At moderate dose, Mlkl^−/− mice are indistinguishable from Wt in terms of survival. At lethal dose, Mlkl^−/− mice show increased survival; fewer disrupted epithelial cells despite same viral titre 6 days p.i. Influx of neutrophils into the lungs in Mlkl^−/− mice both delayed and diminished.	Sex-matched littermates used for Mlkl^−/− mice [5].	[13]
	Instranasal 2500 EID₅₀. Instranasal 1500 EID₅₀.	Mlkl^−/− are indistinguishable from Wt controls in terms of survival, viral titres, morphometry of viral spread and percentage of infected lung. IAV-mediated alveolar inflammation; septal thickening, inflamed alveoli, and hyaline membranes in Mlkl^−/− mice comparable to that of Wt mice.	Sex matched Wt littermate or Wt C57BL/6 mice were used as controls for Mlkl^−/− mice [5].	[51]
Influenza strain A/California/7/2009	250 PFU of IAV.	At baseline, Mlkl^−/− mouse myocardium showed increased mitochondrial and antioxidant activity (proteome analysis), increased survival and reduced weight loss.	Wt (B6NTac) mice and Mlkl^−/− mice [5] were bred independently.	[52]
Secondary S. pneumoniae following influenza A viral infection	Intranasal 250 PFU pdmH1N1. Intratracheal 1 × 10³ CFU of S. pneumoniae.	Mlkl^−/− mice showed reduced pulmonary cell death (TUNEL-staining), bacterial burden, lung consolidation and IFN-α, -β expression. No changes in the amount of oxidative-stress-induced DNA damage (immunofluorescence of 8-OHdG).	Wt and Mlkl^−/− [5].	[53]
West Nile Virus (WNV) encephalitis	Subcutaneous injection with 100 pfu of WNV-TX 2002-HC strain.	Mlkl^−/− mice are indistinguishable from Wt in terms of survival and viral titres.	Sex matched Wt C57BL/6J used for Mlkl^−/− mice [5].	[54]
Metabolic
Non-alcoholic fatty liver disease	Choline-deficient, methionine-supplemented (CDE), fed once.	Mlkl^−/− mice were indistinguishable from Wt controls in terms of hepatic necrosis (serum AST and PI-positive cells). Reduced systemic levels of Il-6 and IL-1β (RT-PCR), comparable levels of TNFα.	Male C57BL/6J (CLEA Japan) including Mlkl^−/− (M.Pasparakis) were utilised.	[55]
	12 weeks of high fat diet.	Mlkl^−/− mice gain body weight comparable to Wt. At baseline, Mlkl^−/− mice had increased serum AST/ALT and decreased serum fasting blood glucose. Mlkl^−/− mice demonstrate reduced NAFLD activity score, steatosis score, hepatocyte ballooning, lobular inflammation, serum AST/ALT, triglyceride levels and de novo fat synthesis.	C57BL/6N Wt and Mlkl^−/− (Jiahuai Han) mice utilised.	[56]
	8 weeks of Western diet.	Mlkl^−/− are indistinguishable from Wt in terms of level of steatosis and liver triglyceride accumulation.	Sex-matched Mlkl^−/− (CRISPR-Cas9) on C57BL/6N and Wt utilised.	[17]
	12 weeks of fat, fructose and cholesterol (FFC) diet.	Mlkl^−/− mice are protected from liver injury (measured using AST/ALT, hepatic triglyceride accumulation, macrovesicular and microvesicular steatosis (H&E). Mlkl^−/− mice were protected from FFC-induced apoptosis (measured using M30 levels, cleaved caspase-3, and TUNEL-positive cells) and inflammation (measured using mRNA levels of TNFα, Il-1β, MCP-1 and F4/80).	Littermates utilised.	[57]
Alcoholic fatty liver disease (ALFD)	Chronic ethanol-induced.	Mlkl^−/− mice are indistinguishable from Wt in terms of ALT/AST, hepatic triglycerides, macrovesicular and microvesicular steatosis (H&E).	Sex-matched littermates utilised.	[58]
Alcoholic fatty liver disease (ALFD)	Gao-binge.	Mlkl^−/− mice are indistinguishable from Wt in terms of body weight, food intake, ALT/AST, hepatic triglycerides, macrovesicular and microvesicular steatosis (H&E). Mlkl^−/− mice have similar levels of CYP2E1, ER stress and hepatocyte apoptosis but mildly reduced levels of some hepatic inflammatory markers.	Sex-matched littermates utilised.	[58]
Diet-induced obesity	16 weeks of high fat diet (HFD) consisting of 60% kcal from fat or chow diet (CD).	Mlkl^−/− mice on regular CD are indistinguishable from Wt littermates in terms of body weight, glucose disposal, glucose tolerance, or insulin sensitivity. After 16 weeks on HFD, Mlkl^−/− mice have lower body weight and visceral adipose, and better glucose and insulin tolerance. No difference in inflammatory markers or TUNEL positive cells in the liver.	Body weight matched, male Wt and Mlkl^−/− littermates are utilised.	[59]
Atherosclerosis	Western diet for 8 or 16 weeks.	Apoe^−/− mice fed a Western diet while receiving MLKL antisense oligonucleotides (ASOs) demonstrated reduced necrotic core size of aortic sinus plaques, reduced plasma cholesterol, fewer TUNEL positive cells but increased lipid content in atherosclerotic plaque (oil red O staining).	Apoe^−/− C57Bl/6N mice administered with control or MLKL antisense oligonucleotides (ASOs).	[60]
Neoplasia
Acute myeloid leukemia	Retroviral expression of the fusion protein (MLL-ENL) in Mlkl^−/− hematopoietic stem cells.	AML cells (MLL-ENL-transduced E14 liver hematopoietic stem cells (HSCs)) transplanted into lethally irradiated Wt mice. AML generated from Mlkl^−/− HSCs showed similar leukemia progression and overall survival compared to AML generated from Wt HSCs.	Age matched, both Mlkl^−/− and Wt on C57B/6J background.	[61]
Colon cancer	Sporadic intestinal adenoma (APC^min mouse).	No significant difference in colonic tumour burden. No difference in colonic inflammation (H&E) or expression of Il-6.	Wt and Mlkl^−/− [5] are littermates. Data for both sexes presented.	[35]
Colon cancer	Colitis-associated cancer (azoxymethane, DSS).	No significant difference in weight loss observed. Similar timing of tumour onset and burden between Mlkl^−/− and Wt (endoscopic tumour scores and H&E).	Wt and Mlkl^−/− [5] are littermates. Data for both sexes presented.	[35]
Colitis-associated tumorigeneses	AOM injection at 10 mg/kg of body weight. Five days post, fed with three cycles of 3% (w/v) DSS, followed by 14 days of normal water.	Mlkl^−/− mice have a significant reduction in body weight, increased clinical severity, shorter colons, and worse survival in comparison to Wt. Mlkl^−/− mice exhibit increased burden of anal and colonic polyps with significant increase in inflammation, hyperplasia, and dysplasia (H&E).	Wt and Mlkl^−/− (CRISPR-Cas9) were sex-matched (males only).	[62]
Colitis-associated tumorigeneses		Apc^min/+ Mlkl^−/− mice have a median survival time of 127 days compared with Apc^min/+ survival of 185 days, alongside marked increases in tumour number and load.	Wt and Mlkl^−/− (CRISPR-Cas9) were littermate controls.	[63]
Progressive lymphoproliferative disease	Fadd gene knock out.	Mlkl^−/− rescues Fadd^−/− mice from embryonic lethality but results in more severe progressive lymphoproliferative disease compared to Ripk3^−/−Fadd^−/− mice.	Mlkl^−/− generated by CRISPR-Cas9 (Bioray Labs).	[64]
	Fadd gene knock out.		Littermates derived from Fadd^+/−, Mlkl^+/− crosses [5] mice utilised.	[65]
	Casp8 gene knock out.	Mlkl^−/− rescues Casp8^−/− mice from embryonic lethality but results in more severe progressive lymphoproliferative disease compared to Ripk3^−/−Casp8^−/− mice.	Littermates derived from Casp8^+/−, Mlkl^+/− crosses [5] mice utilised.	[65]
Neuromuscular
Amyotrophic lateral sclerosis	SOD1^G93A mice (express mutant human superoxide dismutase 1).	MLKL deficiency does not affect disease onset, progression, or survival in SOD1^G93A mice. MLKL ablation has no impact on astrocyte or microglial activation.	Four isogenic genotypes for study: SOD1^G93A;Mlkl^−/−, SOD1^G93A, Mlkl^−/− and Wt littermates. Mlkl^−/− mice [5].	[66]
Sciatic nerve crush injury	Sciatic nerve cut or crushed unilaterally.	pMLKL (serine 441) is upregulated in damaged sciatic nerve cells of both Wt and Ripk3^−/− mouse sciatic nerves. Mlkl^−/− mice suffer drastically reduced myelin sheath breakdown and thus reduced sciatic nerve regeneration and function.	Male only Mlkl^−/− (Li et al., 2017) and Wt (C57BL/6J) utilised.	[67]
Experimental autoimmune encephalomyelitis (EAE)	Intraperitoneal injection of 200ng of pertussis toxin.	Mlkl^−/− mice display significantly reduced clinical EAE disease scores with delayed reduction of myelination (MBP immunofluorescence).	Sex matched Wt and Mlkl^−/− (CRISPR-Cas9) bred as separate cohorts. Same housing conditions since birth.	[68]
Cuprizone CPZ-induced demyelination	Fed 0.2% CPZ in chow for 4 weeks.	Mlkl^−/− mice have delayed demyelination in the caudal corpus callosum (MBP immunofluorescence).	Sex matched Wt and Mlkl^−/− (CRISPR-Cas9) bred as separate cohorts. Same housing conditions since birth.	[68]
Parkinson’s disease (PD)	Intraperitoneal injection of MPTP.	Mlkl^−/− show reduced neuroinflammatory markers (TNFα, IL-1β and IL-1 mRNA) and are significantly protected from striatal dopamine reduction and TH-positive neuron loss from the substantia nigra pars compacta compared to Wt controls.	Male Mlkl^−/− (Jiahuai Han, Xiamen University, China) and Wt utilised.	[69]
Acute muscle injury	Intramuscular injection of cardiotoxin (CTX).	Mlkl^−/− mice have fewer active muscle stem cells and thus suffer a significantly impaired capacity to regenerate muscle fibres. Regenerating myofibrils from Mlkl^−/− mice express less myogenic factors MyoD, MyoG and nascent myofibril marker MYH3 in contrast to Wt.	Male Mlkl^−/− (CRISPR-Cas9) and Wt mice utilised.	[70]
Hematological
Venous thromboembolic (VTE) disease	100% flow obstruction of the IVC using ETHICON.	Mlkl^−/− mice develop significantly smaller thrombi, with reduced areas of TUNEL+ cells, Ly6b+ neutrophils and F4/80+ macrophages. Systemically, Mlkl^−/− mice have fewer circulating neutrophils, monocytes, and serum histone–DNA complexes following IVC ligation. No difference in baseline number of neutrophils, monocytes, DAMPs or bleeding time.	Male Wt (C57BL/6N) and Mlkl^−/− [5].	[71]
Baseline	Mlkl^−/− mice aged to 50 and 100 days.	Mlkl^−/− mice are indistinguishable from Wt for the following blood parameters: WBC and lymphocyte count, thymic weight and cell count, splenic weight and cell count, lymph node weight and cell count.	Littermates derived from Casp8^+/−, Mlkl^+/− crosses [5] mice utilised.	[65]
Casp8 or FADD deficiency	Casp8 or FADD deficient background.	Compared with Casp8^−/−Ripk3^−/− or Fadd^−/−Ripk3^−/− mice, Casp8^−/−Mlkl^−/− or Fadd^−/−Mlkl^−/− demonstrate more severe lymphadenopathy and autoimmune manifestations.	Littermates derived from Casp8^+/−, Mlkl^+/− crosses [5] mice utilised.	[65]
Reconstitution of the hematopoietic system	Competitive transplantation assay using myeloablated recipients.	Following myeloablation, Mlkl^−/− bone marrow stem cells were able to compete effectively with Wt counterparts for reconstitution of the hematopoietic system (blood, bone marrow and spleen).	Littermate controls.	[5]
Mlkl^D139V-induced hematopoietic defects	Whole body homozygosity for constitutively active Mlkl^D139V mutation.	P3 Mlkl^D139V/D139V mice had significant deficits in lymphocyte and platelet counts in comparison to P3 Mlkl^WT/WT and Mlkl^D139V/WT.	Age and sex matched littermate controls.	[40]
	Myelosuppressive irradiation.	Recovery was delayed in Mlkl^WT/D139V adult mice.	Age and sex matched littermate controls.	[40]
	5-fluorouracil.	Adult Mlkl^D139V/WT mice had delayed recovery of hematopoietic stem and progenitor cells.	Age and sex matched littermate controls.	[40]
	Competitive bone marrow transplants.	Bone marrow-derived HSCs from Mlkl^WT/D139V adults and foetal liver-derived HSCs from Mlkl^WT/D139V and Mlkl^D139V/D139V competed poorly or not at all with co-transplanted Wt bone marrow.	Age and sex matched littermate controls.	[40]
Acute thrombocytopaenia	Single dose of anti-platelet serum.	Mlkl^−/− mounted a similar recovery to Wt in terms of magnitude and kinetics.	Wt and Mlkl^−/− [5]	[72]
Hemostasis	Bleed times into 37 °C saline, after 3-mm tail amputations, measured over 10 min.	Mlkl^−/− show prolonged bleeding times compared to Wt and yet equivalent total blood loss.	Wt and Mlkl^−/− [5]	[72]
Reproductive system
Male reproductive system aging	Male mice aged 15 months.	Mlkl^−/− mice showed reduced body weight, reduced seminal vesicle weight, increased testosterone, increased fertility and fewer empty seminiferous tubules.	Wt and Mlkl^−/− (CRISPR-Cas9) bred as separate cohorts. Same housing conditions since birth.	[73]
Male reproductive system aging	Male mice aged 18 months.	No significant difference in mortality, body weight, testis weight, seminal vesicle weight or germ cell loss in seminiferous tubules between Mlkl^−/− mice and Wt mice.	Wt and Mlkl^−/− [5] are litter-mate controls.	[74]
Other
Ventilator-induced lung injury (VILI)		Mlkl^−/− mice were not protected against VILI at low or high tidal volumes.	Littermate Wt used for Mlkl^−/− (Jiahuai Han Laboratory) mice.	[75]
Progressive renal fibrosis	Left urethral obstruction (UUO) by double ligation.	Mlkl^−/− mice were not protected from renal damage.	Sex-matched male Wt and Mlkl^−/− (Jiahuai Han Laboratory) mice.	[76]

* Methods not available.