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. 2017 Jul 16;2017:3464953. doi: 10.1155/2017/3464953

Figure 3.

Figure 3

TSA coaddition with BIX01294 promotes greater responsiveness to the cardiogenic stimulating factor Wnt11. MSCs were cultured in the absence or presence of 8 μM BIX01294 plus or minus either 50 nM TSA or 1 μM Npy19 for 48 hrs, prior to seven-day culture in fresh media with or without Wnt11. (a)–(c) Real-time qPCR analysis of RNA harvested from the cultures indicates that the primary cardiac transcription factors Nkx2.5, GATA4, and myocardin were significantly upregulated in MSCs in response to Wnt11, but only when pretreated with BIX01294. When TSA (but not Npy19) was combined with BIX01294 during the initial 48 hr incubation period, a significant enhancement was observed for Nkx2.5 (n = 11), GATA4 (n = 11), and myocardin (n = 8) of 4.3-, 2.6-, and 5.6-fold, respectively. Statistical significance is shown for comparative levels of Nkx2.5, GATA4, or myocardin expression displayed by Wnt11 stimulated cultures pretreated with either TSA plus BIX01294 or BIX01294, and nonpretreatment controls (∗p < 0.05; ∗∗p < 0.01). (d) Identification of individual sarcomeric α-actinin positive cells (arrows) by immunofluorescent staining indicated that cardiac protein expression within the MSC cultures was in accordance to the gene expression patterns, as shown in this panel for cultures treated sequentially with TSA plus BIX01294 followed by Wnt11. Scale bar = 25 μm. (e) Tabulation of α-actinin positive cells within these cultures demonstrated that BIX01294 or BIX01294+TSA pretreatments significantly increased the number of cardiac protein expressing cells as compared to nontreated or Wnt11 only conditions. Statistical significance is indicated by ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.