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. 2017 Jun 23;18(7):1348. doi: 10.3390/ijms18071348

Figure 1.

Figure 1

BMP9 enhanced the expression level of RUNX1 in MSCs and MMCs, RUNX1 was a direct target of BMP9/Smad signaling. (A,B) The gene expression level of RUNX1 was detected by RT-PCR in C3H10T1/2, C2C12 and MEFs after Ad-BMP9 infection for 48 h. (C,D) The protein expression level of RUNX1 was detected by Western blotin C3H10T1/2, C2C12 and MEFs after Ad-BMP9 infection for 72 h. (E) A schematic presentation of the 2.0 kb promoter region of mouse RUNX1. (F,G) ChIP analysis of the mouse RUNX1 promoter. MEFs cells were infected with Ad-GFP or Ad-BMP9. Cells were cross-linked. The cross-linked cells were lysed and subjected to sonication, following immunoprecipitation with Smad1/5/8 antibody or IgG. The retrieved genomic DNA was amplified by RT-RCR using the two pairs of primers, PP-1 and PP-2. The genomic DNA input was comparable among the samples. (H,I) the protein expression level of RUNX1 was detected by Western blot in C3H10T1/2, C2C12 and MEFs after Ad-RUNX1 infection for 72 h. (J) The protein expression level of RUNX1 was detected by Western blot in C3H10T1/2, C2C12 and MEFs. (K,L) The protein expression level of RUNX1 was detected by Western blot in C2C12 and MEFs after Ad-siRUNX1 infection for 72 h. GAPDH and β-actin were used as loading control, separately. * p < 0.05, as compared with control group. RUNX: runt-related transcription factor; MSCs: Mesenchymal stem cells; MMCs: murine multi-lineage cells; RT-PCR: Semi-Quantitative PCR; ChIP: Chromatin Immunoprecipitation; Ad-GFP: Adenovirus carrying green fluorescent protein gene; Ad-BMP9: :Adenovirus carrying bone morphogenetic protein 9 gene; IgG: Immunoglobulin G; GAPDH: Glyceraldehyde 3-phosphate-dehydrogenase.