Additional file 1 of BET protein inhibitor apabetalone (RVX-208) suppresses pro-inflammatory hyper-activation of monocytes from patients with cardiovascular disease and type 2 diabetes

Additional file 1–3. Additional file 1. Analysis of monocyte subpopulations in control (CTL) and DM2+CVD patients. Monocytes were classified as classical (CD14++CD16−), intermediate (CD14++CD16+) or non-classical (CD14+CD16+). Fluorescence was measured using a FACS CANTO II (BD) and analysed with FlowJo software. Additional file 2. Monocytes were stimulated ex vivo with IFNγ (1.5, 3.12, 6.25, 12.5 or 25 U/ml), apabetalone (1.5, 3.12, 6.25, 12.5 or 25 μM), or a combination of both stimuli for 24h. Subsequently, cytotoxicity was determined by measuring the enzyme lactate dehydrogenase (LDH) in the supernatant using the CytoTox 96® non-radioactive cytotoxicity assay (Promega). There was no difference between ‘unstimulated’ and ‘stimulated’ conditions, except for the positive control where p < 0.0001. Statistics: One-way ANOVA with Dunnett’s multiple comparisons test. Additional file 3. BRD4 mRNA expression is reduced by ex vivo treatment with apabetalone in DM2+CVD monocytes (4h ex vivo treatment, 25 μM). BRD4 expression was measured by real-time PCR and normalized to cyclophilin A (endogenous control). Statistics: Unpaired Student’s t-test, ****, p  < 0.0001.