To ascertain the effect of parthenolide and DMAPT on the induction of necrosis, cells were treated with PI, a cell-impermeable nuclear dye, which stains the nuclei of cells that have lost plasma membrane integrity and are considered necrotic.
In parallel, we stained dying cells with the cell-impermeable red fluorescent nuclear dye propidium iodide (PI).
Because the nuclear membrane is impermeable to large molecules, nuclear pores are required that regulate nuclear transport of molecules across the envelope.
Dead cells were identified as SYTOX-green positive due to nuclear staining of impermeable dye.
No swelling of the nucleus is due to permeabilization of the NPC to naturally impermeable macromolecules, e.g., PVP and large nuclear proteins (see the next section for experimental confirmation), which results in an isotonic condition across the NE.
While the intracellular dynamics of PEI-nucleotide complexes once released from the endosome are unknown, oligonucleotides must dissociate from PEI to reach the nucleus, as the nuclear envelope is likely impermeable to PEI.
Neuronal damage was characterized by four different approaches using either Hoechst 33342 (nuclear stain), the membrane-impermeable dye propidium iodide (to detect membrane damage), an antibody against the active (cleaved) form of caspase-3 or the TUNEL assay.
