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
posted on 2020-11-11, 04:23authored bySylwia Wasiak, Kim E. Dzobo, Brooke D. Rakai, Yannick Kaiser, Miranda Versloot, Mahnoush Bahjat, Stephanie C. Stotz, Li Fu, Michael Sweeney, Jan O. Johansson, Norman C. W. Wong, Erik S. G. Stroes, Jeffrey Kroon, Ewelina Kulikowski
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.
Funding
Nederlandse Organisatie voor Wetenschappelijk Onderzoek Hartstichting