Extended Data Fig. 3: Acer1 is downregulated in Platelets from AAA mice and patients.
a-b. Quantitative measurement of Sphingosine (Sph) by mass spectrometry (a) and S1P by ELISA (b) in platelets isolated from mice infused with saline or AngII (1000 ng/kg/min) for 7 days. n = 5 mice per group. c. Flow cytometric analysis of Acer1 expression in megakarocytes from C57BL6 mice infused with saline or AngII. n = 5 mice per group. d. Western blot analysis of the half-life (t1/2) of Acer1 protein in mouse platelets pretreated with cycloheximide (CHX, 10 μg/mL) for 30 min to inhibit translation, followed with saline (as vehicle) or AngII (1 μM) treatment for different time periods. The relative level of Acer1 expression at the 0 h time point was set as 100%. n = 3 biologically independent experiments. e-f. qRT-PCR (e) and Western blot analysis (f) of Acer1 expression in mouse platelets stimulated with AngII (1 μM) or saline as vehicle for 6 hours in vitro. n = 4 biologically independent samples. g. Alkaline ceramidase activity of mouse platelets stimulated with AngII (1 μM) for 6 hours in vitro. n = 8 biologically independent samples. h. Left: Western blot analysis of Meg-01 cells-produced PLPs transfected with pcDNA3.1 and pcDNA3.1-Acer1 plasmids. Right: Alkaline ceramidase activity of PLPs with or without Acer1 overexpresion. n = 4 biologically independent experiments. i. Alkaline ceramidase activity of platelets from Acer1-/- and littermate WT mice infused with saline or AngII (1000 ng/kg/min) for 7 days. n = 5 biologically independent samples. j-m. qRT-PCR evaluation of enzyme genes of ceramide metabolism pathways, including the de novo pathway (j), sphingomyelinase pathway (k), salvage pathway (l) and hydrolysis pathway (m), in platelets from AAA patients and control individuals (non-AAA). n = 5 biologically independent samples. n. Quantitative measurement of ceramides by mass spectrometry in circulating platelets from Acer1-/- and littermate C57BL6 mice infused with AngII (1000 ng/kg/min) for 7 days. n = 5 mice per group. Data are presented as mean ± SEM. P values were calculated by the two-sided Mann-Whitney test (a-c, e-h, j-n), the two-sided unpaired Student’s t test (d) or ANOVA followed by the Bonferroni test (i).
