HTRF Total MLKL Cellular Kit HTRF®

The human colorectal cancer cell line HT-29 was seeded in a 96-well culture-treated plate under 200,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. For MLKL activation experiments, the cells were pre-treated with 25 µM Z-VAD (pan-caspase inhibitor) for 30 minutes before the simultaneous addition of SM-164 (cIAP1/2 inhibitor) at 100 nM and human TNF-α at various concentrations for 6 hours. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-MLKL (Ser358) or Total-MLKL detection reagents were added. The HTRF signal was recorded after an overnight incubation.

In presence of SM-164 and Z-VAD, that block cell survival and apoptosis respectively, TNF-α induces a dose-dependent increase in MLKL phosphorylation at Ser358, highlighting the activation of the necroptosis pathway. Total MLKL show unchanged levels of signal, demonstrating that the modulations observed on Phospho-MLKL is induced by the pharmacological treatments, and is not caused by cell detachment that can happen during late stages of necroptosis.

The human colorectal cancer cell line HT-29 was seeded in a 96-well culture-treated plate under 200,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. The cells were pre-treated with 25 µM Z-VAD for 20 minutes before the addition of increasing doses of Necrostatin-1s or Necrostatin-1. After 10 additional minutes, a pre-mix containing 100 ng/mL TNF-α and 100 nM SM-164 was added for 6h. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Phospho-MLK (Ser358) or Total-MLKL detection reagents were added. The HTRF signal was recorded after an overnight incubation.

The necroptosis inhibitor Necrostatin-1 and its analog Necrostatin-1s both induce a dose-dependent decrease in MLKL phosphorylation at Ser358, while total MLKL remain stable. As expected, Necrostatin-1s is more potent than the original small molecule, based on the IC50 values obtained on Phospho-MLKL that are ~ 2 times better than with Necrostatin-1.

The human colorectal cancer cell line HT-29 was cultured in a T175 flask in complete culture medium for 48h at 37°C, 5% CO2. The cells were pre-treated with 25 µM Z-VAD for 30 minutes before the simultaneous addition of SM-164 at 100 nM and human TNF-α at 100 ng/mL for 6 hours. After treatment, the cells were lyzed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total MLKL detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF Total MLKL assay, 4,100 cells/well were enough to detect a significant signal, while 8,125 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Total MLKL assay was 2 times more sensitive than the Western Blot technique.

MLKL plays key role in TNF-a induced necroptosis, programmed cell death process.

Upon binding of TNF-α to TNFR1, the receptor triggers the rapid formation of complex I at the cytoplasmic membrane by recruiting multiple proteins, including RIPK1, TRADD, TRAF2, and the cellular inhibitors of apoptosis 1 and 2 (cIAP1/2). cIAP1/2 catalyze the polyubiquitination of RIPK1, leading to the recruitment of IKKα and IKKβ and the activation of the NF-κB pro-survival pathway.

In the absence of cIAP1/2, RIPK1 is released from TNFR1 and associates with FADD and caspase 8 to form the cytosolic complex IIa, responsible for caspase 8-dependent cell apoptosis.

When caspase 8 activity is inhibited or under pathological conditions, MLKL interacts with RIPK1 and RIPK3 to form the cytosolic complex IIb (also called 'necrosome') which is involved in the initiation of necroptosis. Following RIPK1 autophosphorylation MLKL is phosphorylated by RIPK3 at Ser358 leading to its oligomerization, localization to the plasma membrane and execution of programmed necrosis characterized by calcium influx and plasma membrane damage . DAMPs are then released to promote damage repairs.