Hoechst 33342

Peptides SS-31, SS-20, SPN4, and SPN10 were prepared by solid-phase synthesis as trifluoroacetic acid (TFA) salts by Phoenix Pharmaceuticals (Burlingame, CA). Powder stocks were reconstituted to a concentration of 10 mM as aqueous solutions and stored at –20°C. Synthetic phospholipids were purchased as chloroform stocks form Avanti Polar Lipids (Alabaster, AL), including POPC (1-palmitoleoyl-2-oleoyl-sn-glycero-3-phosphocholine), DHPC (1,2-diheptanoyl-sn-glycero-3-phosphocholine), and TOCL (1´,3´-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol). All lipid stocks were stored at –20°C in clear glass vials with teflon-lined cap closures. For analytical spectroscopy, fluorescent probes included 1,8-ANS, di-ANEPPS, and TMRM (Thermo Fisher, Waltham, MA). For microscopy, fluorescent probes included TMRM and MitoView Green (Biotium, Fremont, CA) and Hoechst 33342 (Novus, Centennial, CO). All solutions were prepared with ultrapure water (Millipore Advantage A10 system; resistivity 18.2 MΩ•cm @ 25 °C; total oxidizable carbon 4 ppb).

After collection, aliquots (100 µL) of A549 media were dried under N₂ gas and prepared in LDS sample buffer for electrophoresis separation and membrane transfer. HMGB1 was detected via an HMGB1 rabbit pAb (ab79823) and antirabbit HRP secondary antibody and visualized with an ImageQuant LAS 4000 camera. HMGB2 was similarly detected via an HMGB2 rabbit pAb (ab124670) and antirabbit HRP. Western blots were developed with ECL reagents. Fluorescent staining for nuclear DNA, necrosis, and apoptosis was done with Hoechst 33342, pSIVA, and PI dyes (Novus, NBP2-29382), respectively, following washing of live cells with PBS. Fluorescent microscopy was performed with BioTek Gen5 3.04 Imager. Images were generated from overlaying the blue, green, and magenta filters, respectively.

Cell uptake of SS-31 and SPN10 was determined using N-biotinylated SS-31 and SPN10 and detected by streptavidin binding. After 3 days serum deprivation to deplete endogenous biotin, cells were treated with 1 µM biotinylated peptides for 1 hr before they were fixed with 4% PFA and incubated with Streptavidin-AlexaFluor 594 antibody (Jackson ImmunoResearch, West Grove, PA) and Hoechst 33342 (Novus Biologicals, Centennial, CO). Images were obtained with a Nikon Eclipse Ti2 fluorescent microscope using a 100× objective.