Fig 2: The Dll4in3 enhancer requires MEF2 binding to direct expression during adult neovascularization. (A) Representative images of Dll4in3:LacZ transgene expression during neovascularization into Matrigel plugs and B16F10 melanoma tumors demonstrate robust vascular X-gal staining. (B) Representative images of the mutant Dll4in3mutMEF:LacZ transgene demonstrate that the mutated enhancer was unable to direct any detectable reporter gene expression during adult neovascularization. See also Supplemental Figure 3.

Fig 3: SIK inhibitors block MEF2C function.(A) Effects of the compounds from the Selleck Epigenetic Compound Library and various additional inhibitors (1 µM) were screened for their potential to induce a CD3e+ phenotype in LOUCY cells on DLL4-coated plates. As controls, media, non-DLL4–stimulated cells or MEF2C-overexpressing (LOUCY_MEF2C-BFP) cells (+dox) have been indicated in blue. Normalized CD3e fluorescence intensities have been plotted for duplicate experiments. (B) SIK kinase inhibitors YKL-06-062 and HG-9-91-01; dasatinib, which has SIK off-target activity (12 nM, 48 nM, and 180 nM for SIK1, SIK2, or SIK3, respectively) (MedChemExpress or Proteomicsdb.org), and imatinib were tested to induce a CD3e+ phenotype in LOUCY_MEF2C-BFP cells when cultured on DLL4-coated plates in the absence of dox (except when indicated). (C) Western blot of MEF2C, P-MEF2C (Ser222), and BCL2 in noninduced (no dox) or MEF2C-induced (+dox) LOUCY_MEF2C-BFP cells that were incubated in the presence of increasing concentrations of inhibitors HG-9-91-01 for 4 days. Band intensity ratios for various proteins relatively to ß-actin control levels is indicated, with the –dox condition set at the value = 1. (D) Cell viability of noninduced (no dox) or MEF2C-induced (+dox) LOUCY_MEF2C-BFP after a 4-day exposure to a serial dilution of prednisolone combined with a serial dilution of HG-9-91-01 at the concentrations indicated. ZIP synergy scores are calculated from an n = 3 per condition. ZIP scores < –10 are indicated with a dash-dot line and a blue color. ZIP scores > 10 are indicated by a dashed line.

Fig 4: Has2os expression was increased in differentiated muscle cells. (A). The morphological changes in C2C12 cells before and after differentiation. D0 represents cells in growth medium, and D1, D3, and D5 represent cells switched into differentiation medium for 1, 3, or 5 days. Scale bar = 200 µm. (B). The mRNA expression levels of the myogenic marker MyHC, Mef2C, MyoD, and MyoG were measured before and after the differentiation of C2C12 cells. (C). The protein expression levels of myogenic markers MyHC and MEF2C were detected by Western blot. GAPDH was the internal control. (D). Relative expression in (C) were calculated. (E). The expression levels of Has2os in D0, D1, D3, and D5. GAPDH was the internal control. Values were presented as means ± SEM. The statistical significance was calculated by t-test.

Fig 5: Effects of pulsed PTHrP treatment on RUNX2, RUNX3 and MEF2C. MPC pellets were subjected to chondrogenic induction for 1 week before half of the pellets were stimulated with 2.5 nM PTHrP(1-34) for 6 h daily while the others received daily medium exchange until week 6 (n = 5 donors). Expression of indicated genes was determined by qPCR (A–C), and levels were referred to reference genes HNRPH1 and CPSF6. (A) Gene expression of the HH downstream target GLI1 (n = 3). ALP activity was quantified in culture supernatants (B). Expression levels of indicated genes and ALP activity are referred to DMSO cultures (dashed lines). Box plots were generated as described in Figure 1, outliers are depicted as ° and extreme values as ?. *, p = 0.05, Mann–Whitney U test compared to control. (D) Overview of pathways regulating RUNX2, RUNX3, and MEF2C during MPC chondrogenesis.