Journal: bioRxiv
Article Title: Multitargeted Reduction of Inflammation and Atherosclerosis in Tet2 -deficient CHIP via XPO1 Inhibition and Atf3 restoration
doi: 10.1101/2025.06.12.658927
Figure Lengend Snippet: a, Macrophages were obtained from the bone marrow of Tet2+/+ , Tet2+/- or Tet2-/- mice and cultured for 6 days in IMDM plus M-CSF followed by incubation for 24 hours with hLDL (200 mg/dl) and either eltanexor (100 nM) or vehicle control. Bone marrow-derived macrophages (BMDM) were then harvested and processed for ChIP-seq, CUT&RUN and RNA-seq experiments. b , Enhancer profiling of BMDM from Tet2+/+ , Tet2+/- or Tet2-/- mice revealed a conserved set of SEs associated with genes encoding transcription factors (n=31). The enhancers are highlighted in red in mouse genotypes when they are large enough to qualify as SEs, and in black when they do not based on the H3K27Ac signal. c , Ranking of enhancers by H3K27ac signal associated with genes in BMDM from Tet2+/+ mice. Atf3 was associated with the largest SE in Tet2+/+ BMDMs. Atf3 binding partners Atf4, Jun, Junb, Jund, and Cebpa are also indicated. d , Normalized ChIP-seq alignment tracks for H3K27ac at the Atf3 -associated SE in BMDM from Tet2+/+, Tet2+/- or Tet2-/- mice. ChIP-seq read densities (y-axis) were normalized to reads per million reads sequenced from each sample. Red bar indicates the location of SEs. e-j , Gene expression levels by RNAseq of Atf3 (e), Cebpa (f) Atf4 (g), Junb (h) Jun (i) Jund (j) in BMDM from Tet2+/+ , Tet2+/- or Tet2-/- mice. k , Genome-wide occupancy for ATF3 from control Tet2+/+ and Tet2-/- BMDM and from eltanexor-treated Tet2-/- BMDM determined by CUT&RUN. Genomic regions (rows) were defined as those enriched in sequencing reads for at least one condition and are ranked by the ATF3 signal across the region. l-m , CUT&RUN coverage tracks for ATF3 and IgG (control) in DMSO control- or Eltanexor (Elta)-treated BMDM from Tet2+/+, Tet2+/- or Tet2-/- mice, overlaid with H3K27ac ChIP-seq at the Il1b locus (l) and the Cxcl12 locus (m).
Article Snippet: Red cell lysis with ACK Lysing Buffer (Gibco Cat. No. 10492-01) was performed and bone marrow was cultured by creating a single-cell suspension of whole bone marrow in Iscove’s Modification of DMEM (IMDM) (Corning Cat. No. 10016CV) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific Cat. No. FB- 11), 10 ng/mL recombinant mouse macrophage colony-stimulating factor (M- CSF, Miltenyi Biotec Cat. No. 130-101-706), and 1% penicillin/streptomycin/glutamine (PSG) (Gibco Cat. No.10378-016) in 30 mL total volume.
Techniques: Cell Culture, Incubation, Control, Derivative Assay, ChIP-sequencing, RNA Sequencing, Binding Assay, Gene Expression, Genome Wide, Sequencing
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