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Fig. 4 | Epigenetics & Chromatin

Fig. 4

From: MMP-2 is a novel histone H3 N-terminal protease necessary for myogenic gene activation

Fig. 4

Nuclear MMP-2 activity is required for H3NT proteolysis and myogenic gene activation. a RT-qPCR analysis of total RNA purified from C2C12 wild type myoblasts (D0 WT) and C2C12 cells expressing an MMP-2-specific shRNA (shMMP-2) cultured in differentiation media or MMP-2-rich conditioned media (+CM) for 4 days. qPCR was performed for MMP-2 and the indicated myogenic genes (x-axis), normalized to the 18S housekeeping gene and plotted relative to C2C12 day 4 wild type control (D4 WT) for each gene (y-axis). Three independent biological replicates were performed to generate standard deviation (error bars). The Student’s t test was used to determine statistical significance between D4 WT control and D4 shMMP-2: p < 10–6(*). b Western analysis to detect the H3cl product in chromatin purified from the C2C12 cells described above. Amido black staining (AB) of membranes was performed to confirm equivalent loading of chromatin between samples. c Representative immunofluorescence microscopy of C2C12 WT and shMMP-2 cells cultured in differentiation media for 4 days. A myosin heavy chain (MyHC) antibody was used to visualize myotube formation (red) and DAPI used to detect nuclei (blue). d Total number of MyHC positive cells (left) and the average number of nuclei/image (right) in the C2C12 WT and shMMP-2 cells were determined by analyzing 9 random fields from 3 independent biological replicates using a 20× objective. The percent of MyHC positive cells that were mononuclear or multinuclear in the C2C12 WT and shMMP-2 cells was also determined (bottom)

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