E-MTAB-9254_2
A PGE2-MEF2A axis enables context-dependent control of inflammatory gene expression
BMDMs or iMACs were generated via differentiation of bone marrow cells or immortalized precursors, respectively, in culture media containing m-CSF for 7 day. Upon differentiation, BMDMs or iMacs were left untreated or stimulated as described below. 1) BMDM WT were stimulated with LPS, PGE2, IL-4, IL-10, LPS+PGE2, LPS+IL-4, LPS+IL-10 for 4 hours. Fragmented chromatin was immuno-precipitated for H3K27Ac (each condition at least in duplicate). These experiments were performed to investigate the cross-antagonism in the concomitant presence of pro-inflammatory (LPS) and anti-inflammatory stimuli (IL-4, IL-10, or PGE2). 2) BMDM WT were stimulated with LPS, PGE2, LPS+PGE2 for either 2 or 4 hours. Fragmented chromatin was immuno-precipitated for IRF1(4h), STAT1 (4h), PU.1 (2h, also for IL-10 and LPS+IL-10 stimulation), NFkB (2h), MEF2A (2h), MEF2D (2h), MEF2C (2h), JUNB (2h) (each condition in single). These experiments were performed to assess TFs occupancy at previously defined PGE2-sensitive and PGE2-resistant enhancers. 3) MEF2A-deficient iMac clones (D7, A7, A8, C7) and MEF2A-proficient (referred to as wild-type) iMac clones (NE, B3 and D10) were generated via CRISPR/Cas9 and stimulated or not with LPS for either 2 or 4 hours: each condition in single. Fragmented chromatin was immuno-precipitated for H3K27Ac (4h) or for PU1 (2h). These experiments were performed to investigate the role of MEF2A on LPS-induced chromatin remodeling.
BMDMs or iMACs were generated via differentiation of bone marrow cells or immortalized precursors, respectively, in culture media containing m-CSF for 7 day. Upon differentiation, BMDMs or iMacs were left untreated or stimulated as described below. 1) BMDM WT were stimulated with LPS, PGE2, IL-4, IL-10, LPS+PGE2, LPS+IL-4, LPS+IL-10 for 4 hours. Fragmented chromatin was immuno-precipitated for H3K27Ac (each condition at least in duplicate). These experiments were performed to investigate the cross-antagonism in the concomitant presence of pro-inflammatory (LPS) and anti-inflammatory stimuli (IL-4, IL-10, or PGE2). 2) BMDM WT were stimulated with LPS, PGE2, LPS+PGE2 for either 2 or 4 hours. Fragmented chromatin was immuno-precipitated for IRF1(4h), STAT1 (4h), PU.1 (2h, also for IL-10 and LPS+IL-10 stimulation), NFkB (2h), MEF2A (2h), MEF2D (2h), MEF2C (2h), JUNB (2h) (each condition in single). These experiments were performed to assess TFs occupancy at previously defined PGE2-sensitive and PGE2-resistant enhancers. 3) MEF2A-deficient iMac clones (D7, A7, A8, C7) and MEF2A-proficient (referred to as wild-type) iMac clones (NE, B3 and D10) were generated via CRISPR/Cas9 and stimulated or not with LPS for either 2 or 4 hours: each condition in single. Fragmented chromatin was immuno-precipitated for H3K27Ac (4h) or for PU1 (2h). These experiments were performed to investigate the role of MEF2A on LPS-induced chromatin remodeling.