Supplementary Figure 2: HDL reduces cellular cholesterol and inhibits pro-inflammatory gene expression.

a, Immortalised-BMDMs were treated with HDL (2 mg/ml) for indicated times and cholesterol measured by mass spectrometry from cell lysates, supernatants or media with HDL. b,c, Immunoblot of BMDMs pre-treated with HDL (2 mg/ml) for 6 h and stimulated for indicated times with P3C (50 ng/ml) (b) and ELISA of IL-6 secretion (c). d, BMDMs were pre-treated with HDL or native HDL (2 mg/ml) for 6 h and stimulated with CpG (100 nM) for 4 h before mRNA expression was measured by qPCR. e, C3H/HeJ mice were injected i.p with 2 mg native HDL or control filtrate 6 h before injection with CpG (20 μg) and D-gal (10 mg), 1 h later hepatic mRNA expression was measured by qPCR (CpG n=10, native HDL+CpG n=9). f, BMDMs were pre-treated for 6 h with HDL before 4 h with CpG (100 nM) and Actinomycin D (5 μg/ml) for the indicated times to asses the half-life of IL-6 transcripts. Data is normalised to 0 min Actinomycin D sample for respective conditions. g, BMDMs were pre-treated for 12 h HDL before CpG for 4 h and cyclohexamide (10 μg/ml) treatment for the indicated times to assess the half life of IL-1β protein (relative to β-actin). a, A representative graph of two individual experiments is presented (mean ±S.D.). b-d, A representative blot (b) and ELISA (mean ±S.D.) (c,d) of three individual experiments is shown. e, Data are presented as mean values ±S.E.M, CpG versus native HDL+CpG *p<0.05, **p<0.01. f, A representative graph from two independent experiments is shown. g, A single immunoblot is shown and densitometric analysis of IL-1β combined from three independent experiments (mean ±S.E.M).