Fig 1: Quantification of nuclear and intercalated disc p‐MLKL levels. (A) The number of phosphorylated MLKL (p‐MLKL)‐positive cardiomyocyte nuclei and the number of total cardiomyocyte nuclei in myocardial sections were counted, and nuclear p‐MLKL level was expressed as the percentage of the number of p‐MLKL‐positive cardiomyocyte nuclei to the number of total cardiomyocyte nuclei. Using the median value, that is, 25%, patients were classified into a high nuclear p‐MLKL level group (H‐nucMLKL) and a low nuclear p‐MLKL level group (L‐nucMLKL). (B) p‐MLKL expression levels in the intercalated discs of cardiomyocytes were analysed in a field (200 × 200 μm) in which cardiomyocytes were sectioned longitudinally. Intercalated disc areas stained with anti‐p‐MLKL antibodies were selected and quantified using BZ‐X analyzer software, and the percentage of the positive area to the total area of cardiomyocytes was calculated. Using the median value, that is, 0.38%, patients were classified into a high intercalated disc p‐MLKL level group (H‐idMLKL) and a low intercalated disc p‐MLKL level group (L‐idMLKL).
Fig 2: (a) Effect of necrosulfonamide (NSA) and mdivi-1 (m) in grade 1 RCC organ cultures treated with R1TNF. R1TNF alone without NSA (no NSA) induced increased levels of TUNEL+mTECs, which were significantly reduced with 50 μM NSA, and to a lesser extent, with 10 μM or 20 μM NSA (arrows). Cultures pretreated with 5 μM NSA showed comparable levels of TUNEL+mTECs as untreated cultures (UT). (b) The percentage of TUNEL+mTECs and (c) pMLKLSer358 expression presented as mean fluorescent intensity (MFI) in similar cultures. ***P<0.0001 versus UT,**P<0.001 versus R1TNF; *P<0.05 versus R1TNF; ¥P<0.05 versus R1TNF (+20 or 10μM); ±P<0.001 versus R1TNF (+20 or 10 μM); ns, not significant. (d) In comparison with UT, which show a rare TUNEL+mTECs, R1TNF alone (without m) induced increased levels of TUNEL+mTECs, significantly reduced by m (10 μM) with no effect by zVAD.fmk (m+z) but a marked reduction by nec-1 (m+n) comparable with cultures pretreated with a combination of zVAD.fmk and nec-1 (m+n+z). (e) The percentage of TUNEL+mTECs and (f and g) the mean fluorescence intensity (MFI) for pDrp1Ser616 and pDrp1Ser637 in similar cultures. ***P<0.0001 versus UT (e), **P<0.01 versus UT (f), *P<0.05 versus R1TNF, ± P<0.05 versus R1TNF+m. (g) *P<0.05 versus UT, ±P<0.5 versus R1TNF, ¥P<0.05 versus R1TNF+m; ns, not significant. Bars=mean±S.E.M.; n=3 independent experiments from six separate organ culture experiments with similar results. (h) Schematic diagram of the consequences of R1TNF-mediated necroptosis in mTEC in RCC; ligation of TNFR1 results in the recruitment of RIPK1, facilitating its interaction with RIPK3, which in turn recruits and phosphorylates MLKL at Ser358 and Drp1 at Ser616 thus causing their co-localization with the mitochondria. A separate process causes a reduction in pDrp1 at ser637. Nec-1 inhibits RIPK1, and NSA inhibits MLKL and mdivi-1 inhibits Drp1 inhibiting cell death
Fig 3: Putative model of CER-mediated necroptosis and its role in the pathogenesis of preeclampsia. CER overload in preeclamptic placentae primes RIP1/RIP3 necrosome assembly. Conditions of reduced caspase-8 activity, as demonstrated in E-PE, favor CER to cause MLKL phosphorylation and oligomerization at the cell membrane where it executes necroptosis by permeabilizing the membrane. Necroptotic cell death also prevents normal cytotrophoblast fusion that is required to replenish the syncytiotrophoblast layer, resulting in improper cell turnover
Fig 4: Flow chart of inclusion of patients and study protocol. To examine the difference in DCM phenotypes and adverse event rates according to p‐MLKL expression levels, patients were classified into subgroups using median p‐MLKL level as follows: a high p‐MLKL level group (H‐MLKL) and a low p‐MLKL level group (L‐MLKL); a high nuclear p‐MLKL level group (H‐nucMLKL) and a low nuclear p‐MLKL level group (L‐nucMLKL); and a high intercalated disc p‐MLKL level group (H‐idMLKL) and a low intercalated disc p‐MLKL level group (L‐idMLKL). Normal myocardial samples from three subjects were used for comparison with samples from DCM patients.
Fig 5: Signs of necro(pto)tic cell death during GC culture, evidenced by LDH assay and Western Blot.a Accumulative LDH levels starting of day 2 of GC culture. LDH levels were examined at day 3, 4 and 5 of GC culture using a colorimetric assay. Each data point represents one sample. One-way ANOVA with Dunett correction was carried out to examine statistical significance (*p < 0.05, n = 8, bars indicate SEM). b–d Representative Western Blots and relative quantification of results of GCs cultured for 1, 3, and 5 days. b pRIP1(S166), RIP1 and β−actin Western Blot. Quantification of band intensity was evaluated and expressed relative to RIP1 for the phosphorylated form and relative to β−actin for RIP1 (n = 3, median). c pRIP3(S227), RIP3 and β−actin Western Blot. Quantification of band intensity was evaluated and expressed relative to RIP3 for the phosphorylated form and relative to β−actin for RIP3 (n = 2, median). d pMLKL(T357/S358), MLKL and β−actin Western Blot. Quantification of band intensity was evaluated and expressed relative to MLKL for the phosphorylated form and relative to β−actin for MLKL (n = 3, median)
Supplier Page from Abcam for Human MLKL (phospho S358) peptide