Assessment of the Activation of Multiple Caspases in Macrophages

Begin with a multi-well plate containing adhered bone marrow-derived macrophages.

Introduce influenza A viruses that fuse with macrophages, releasing ribonucleoproteins containing viral RNA.

The macrophages' Z-DNA binding protein 1, or ZBP1 interacts with viral ribonucleoproteins, triggering ZBP1 oligomerization, with kinases and caspase 8.

The oligomerized ZBP1 further recruits inflammasome sensor proteins, adaptor proteins, and procaspase-1, forming a multimeric complex — a PANoptosome.

Within the PANoptosome, initiator caspase-8 activates through proximity cleavage, further activating effector caspases, including caspase-3 and 7.

Additionally, the inflammasome sensor proteins within the PANoptosome activate a signaling cascade, converting pro-caspase-1 to its active form, caspase-1.

Concurrently, viral infection induces the release of cytochrome-c from the mitochondria, triggering apoptosome activation, and converting pro-caspase-9 to its active form.

Viral infection also triggers the non-canonical inflammasome pathway, activating caspase-11.

Introduce a lysis buffer to lyse the macrophages; collect the lysate, and heat it to inactivate proteases, preventing caspases' degradation.

Centrifuge and collect the supernatant containing multiple caspases for further analysis.