Fig 2: Differential interaction of SWI/SNF and Mediator complexes with R3 methylated and unmethylated LAP*/C/EBPß1 N-terminal peptides. (A) The N-terminal sequence of LAP*/C/EBPß1 was synthesized with R3 in unmodified form (1–41) or in asymmetrically dimethylated form (1–41 Rme2a). Peptides with covalently attached C-terminal biotin moieties (grey pentagons) were applied to UNIPEX proteomic libraries and interactions were revealed by streptavidin-HRP (SA-HRP) and ECL. Positive clones were identified as characteristic duplicate pattern per square. Libraries contained 48x48 squares with four duplicate clones on two PVDF membranes. A single square containing the brama-related gene 1 (Brg1) expression clone interacts with unmethylated, but not with methylated peptide (arrow heads). (B) Unmethylated and methylated C/EBPß peptides (1 µM), as shown in (A) were incubated with cell lysates and bound proteins separated by streptavidin Dynabeads. Cell lysates were prepared from Raji cells (top) or HEK-293 MED23-HA-transfected cell lysates (bottom). Western blots were incubated with anti-hBrm, anti-BAF155, anti-BAF47/Ini1, anti-HA, and anti-MED26 as indicated.

Fig 3: CREB-C/EBPß mediates progesterone induction on laminin A5 expression. (A) Western blot analysis of effects of RU486 on progesterone induction on p-CREB and p-C/EBPß-LAP expression in ovariectomized mouse uteri. (B) Western blot analysis of effects of RU486 on progesterone induction on p-CREB and p-C/EBPß-LAP expression in cultured stromal cells. (C) RT-qPCR analysis on effects of CREB inhibitor KG-501 on progesterone induction of Laminin A5 expression in stromal cells. (D) Western blot analysis on effects of KG-501 on progesterone induction of laminin A5, p-CREB, and p-C/EBPß-LAP expression. (E) Western blot analysis on the effect of C/EBPß-LIP overexpression on progesterone induction of laminin A5 expression. (F) RT-qPCR analysis of C/EBPß mRNA levels after stromal cells were treated with C/EBPß siRNA. (G) RT-qPCR analysis of effects of C/EBPß siRNA on progesterone induction of Laminin A5 expression. (H) Western blot analysis of effects of C/EBPß siRNA on progesterone induction of laminin A5, p-CREB, p-C/EBPß-LAP expression. Bars represent mean ± SD (* p-value < 0.05, **** p-value < 0.0001). 3.1, empty pcDNA 3.1 (+) vector; LIP, C/EBPß-LIP overexpression plasmid.

Fig 4: Prep1 down-regulation increases C/EBPß binding to chromatin in 3T3-L1 cells without affecting C/EBPß level and phosphorylation. (A) Immunoblotting analysis of cell extracts from P and C cells at various time points after induction using anti-phospho-C/EBPß (Thr235), and total C/EBPß Abs, recognizing three isoforms of C/EBPß - LAP*, LAP, LIP. Vinculin Ab was used as loading control. (B) Venn diagrams of C/EBPß binding peaks in Prep1 down-regulated (P) and control (C) cells before (-2 d) and 4 hours (4 h) after the induction of differentiation. (C) Venn diagrams and Gene Ontology analysis of genes bound by C/EBPß in P and C cells before (-2 d) and 4 hours (4 h) after induction. (D) DNA-binding motifs (Cebpß and AP1) of C/EBPß binding sites in P and C cells before (-2 d) the induction of differentiation, depicting common and exclusive peaks.

Fig 5: C/EBPß is a mediator of CREB3L4 downregulating the expression of PPAR?2. (a) Oil-red O staining of adipocytes was performed at D8, and microscopy ( × 20) was performed. 3T3-L1 cells were transfected with Cebpb or Cebpb/Creb3l4 and stimulated to differentiate with insulin (1 µg/ml)-containing media (10% FBS-DMEM) every 2 days. (b) Immunoblot of adipogenic transcription factors in 3T3-L1 cells expressing Cebpb or Cebpb/Creb3l4 at D0. ChIP assay performed in 3T3-L1 cells that transfected with siCreb3l4 at D0. Normal IgG was used as a negative control for immunoprecipitation. The regions amplified were -435 to -183 bp for Cebpa gene promoter (c) and -493 to -215 for Pparg2 gene promoter (d). The quantity of immunoprecipitated DNA was normalized to total input DNA. Values are expressed as mean±S.E.M., n=3 per group, *P<0.05. (e) Oil-red O staining of 3T3-L1 cells transfected with siCebpb and siCreb3l4 at D8, and microscopy ( × 20) was performed. The cells were stimulated to differentiate as described in (a). (f) Immunoblot of adipogenic factors in 3T3-L1 cells expressing siCebpb and siCreb3l4, at D0