Animals – Male C57BL/6J (Wildtype, Wt, stock # 000664), homozygous SCD mice (SCD, stock # 013071, Townes model) and PCSK9−/− (stock # 005993) mice were purchased from Jackson Labs (Bar Harbor ME). Bone marrow was transplanted from SCD donor to PCSK9 deficient or WT recipient to generate the experimental PCSK9−/−SCD bone marrow transplant (bmt) mice and the PCSK9+/+SCD bmt controls, as previously [19]. Mice were housed in ventilated cages, on a 12-hour light/dark cycle with food and water given ad libitum. Food, water, and health checks were performed daily by the University of Michigan Unit for Laboratory Medicine certified animal technicians. Sample size was determined by power analysis. All animal use protocols complied with the Principles of Laboratory and Animal Care established by the National Society for Medical Research and were approved by the University of Michigan Institutional Committee on Use and Care of Animals. This study is reported in accordance with ARRIVE guidelines 2.0 from the National Centre for the Replacement Refinement and Reduction of Animals in Research.
Lipidomics – Blood was drawn from the retro-orbital sinus of isoflourane-anesthetized mice (n = 3 per group) into 3.2% sodium citrate. Pelleted erythrocytes were washed 3x with PBS and kept at -80ºC until analyzed by untargeted LC-MS Based Shotgun lipidomics by the Michigan Regional Comprehensive Metabolomics Resource Core. In short, lipids were extracted from samples using a modified Bligh-Dyer method [32] analyzed by reversed-phase high performance liquid chromatography, followed by mass spectrometry analysis. Lipids were identified using the LipidBlast library [33] and quantified using Multiquant, then normalized by internal standards.
Amiodarone treatment – Amiodarone (10 mg/kg) (Hikma Farmaceutica, Portugal) or PBS (Gibco, pH 7.4) was injected daily (intraperitoneal, i.p.) into 8 week old homozygous SCD mice (SCD, stock # 013071, Townes model) for 2 weeks (n = 5 for Amiodarone and n = 4 for PBS). Mice were randomly assigned to each treatment based on cage.
Complete blood counts – Blood samples were withdrawn from the retro-orbital venous plexus into EDTA-lined polythene tubes and were analyzed using a Hemavet 950 (Drew Scientific, Inc). An aliquot of whole blood was mixed with new methylene blue (Ricca Chemical Company) for 20 minutes to stain reticulocytes.
Ex vivo sickling assays – For in vitro experiments, SCD mice (n = 4) were anesthetized using isofluorane, then blood was drawn from the retro-orbital venous plexus into EDTA-lined polythene tubes. SCD RBCs were washed 2x with PBS then resuspended in PBS (30% HCT) and incubated with 50 ug/mL control IgG (Santa Cruz, # SC2025) or BMP antibody (6C4, END Millipore #MABT837) for 4 hours at room temperature. Some erythrocytes were also incubated with 1.5 uM Amiodarone (Hikma Farmaceutica, Portugal) or 6.75 uM MedroxyPROGESTERone Acetate (Amphastar Pharmaceuticals Inc, USA) for 4 hours. A 5 uL aliquot of blood was then placed on a slide with 5 uL sodium metabisulfite (Na2S2O5, Sigma, St. Louis, MO; 2% w/v in PBS), a cover slip applied, then 3 images per slide were captured at 20x after a 60 minute incubation at room temperature using a Nikon SE upright microscope and a Nikon DS-Fi3 camera. The percentage of sickled erythrocytes per image was quantified. Sodium metabisulfite is a reducing agent which scavenges oxygen, promoting deoxygenation in sickle cells [25, 26]. For in vivo treatments, amiodarone- and vehicle-treated SCD mice were anesthetized using isoflurane, then blood was drawn from the retro-orbital venous plexus into EDTA-lined polythene tubes. Sickling percentages were determined in the same manner as in the whole blood sickling assays, described above.
Fluorescence microscopy – Blood samples were withdrawn from the retro-orbital venous plexus into 3.2% sodium citrate, then centrifuged at 105 x g for 5 minutes. The pellet was resuspended in PBS and then fixed for 20 min in 10% formalin + 0.2% glutaraldehyde. At the end of the incubation, cells were centrifuged at 1000 rpm for 5 minutes and then the pellet was resuspended in PBS. Fixed erythrocytes were permeabilized in 0.2% Triton + 3% BSA in PBS for 20 minutes. Cells were resuspended in PBS containing 3% BSA, 5 ug/mL anti-BMP antibody (6C4, END Millipore #MABT837), and 5 ug/mL anti-BAND3 antibody (Invitrogen, #PA5-80030), then incubated for 1 hour at room temperature. Cells were washed 3x with PBS then resuspended in anti-mouse FITC (Abcam, #ab6785; total conc = 2mg/mL) and anti-rabbit Alexafluor-647 (LifeTechnologies, #A21446), then incubated for 1 hour at room temperature while protected from light. After 3 washes, cell suspensions were placed on a glass slide containing Vectashield mounting medium (Vector Laboratories, #H-1400) and a glass coverslip was applied immediately. Three random fields of view at 20x were imaged per sample and analysis was conducted with NIS-Elements (Nikon), using regions of interest to quantify the mean signal intensity for both BMP and BAND3 for each cell imaged.
Statistical analysis – Data are represented as mean ± standard deviation. Statistical analysis was carried out using Graphpad Prism. Normality was determined with a Shapiro-Wilk Test. Significance was determined with a paired student’s t-test. For analysis of multiple groups, significance was determined by an independent a one-way ANOVA, followed by a post-hoc analysis with Turkey’s multiple comparisons tests. Probability values of p < 0.05 were considered statistically significant.