Spermine nonoate

In vitro gene expression of ssaG, fnr, and phoP was assessed using qRT-PCR in bacteria cultured in N-minimal medium with low Mg²⁺ concentration (8 µM MgCl₂) and/or SPI-2 non-inducing (1 mM MgCl₂) N-minimal medium as described above. Gene expression was also assessed in bacteria cultured in N-minimal medium containing 8 µM or 1 mM MgCl₂ under either low or high O₂ conditions. Spermine NONOate (Abcam) was used as the NO donor, and the effect of NO on target gene transcription was investigated. The NO donor was added to the culture after 1 h of bacterial growth in 8 µM or 1 mM MgCl₂ N-minimal medium, when the bacterial cells were in the late log phase.

MK-801 (Hello Bio Inc., 304 Wall Street, Princeton, NJ 08540, USA) and scopolamine (Tocris Bioscience/Biotechne Corporation, Minneapolis, MN, USA) were dissolved in 0.9% saline. Spermine NONOate and DETA NONOate (Abcam, Cambridge, UK) were dissolved in 0.9% saline, and NPLA was dissolved in a small amount of DMSO and then adjusted to the proper volume with 0.9% saline. When the administration of experimental compounds was omitted (control, MK-801, or scopolamine group), the animals received appropriate vehicles. The doses used in behavioural experiments were based on our previous studies [8 (link)].

For competition with unlabeled HDL, a 40-fold excess of HDL (by mass) was added along with 10 μg/mL DiI-HDL or 20 μg/mL AF568-HDL. To inhibit dynamin and caveolae, HCCMECs were treated with 30 μM Dyngo 4a (Abcam, Cambridge, MA) or 50 μg/mL Nystatin (Bioshop Canada, Burlington, ON) in HPMI at 37°C for 30 min prior to the addition of labeled HDL. Nitric oxide donor spermine NONOate (40 μM) (Abcam, Cambridge, MA) or endothelial nitric oxide synthase inhibitor L-NNA (1.6 μM) (Sigma-Aldrich) made up in serum-free media were added to the cells 3 h prior to the addition of labeled HDL. For all pre-treatments, the inhibitors used were re-added during the 10 min internalization step.