Fig 5. Nacα and Hox genes interact to redirect differentiation of Multipotent Cardiac Progenitors (MCPs).

A,C,E, Quantitation of differentiated cell populations 9 days after siRNA treatment. B,D,F. Representative images of immunohistological staining for select conditions. A,B, Total Cell Populations following siRNA treatment were not significantly changed compared to controls, except for Gata4/6,MyoCD siRNA condition which reduced overall cell count. Knockdown (KD) of Nacα (green), HOXC12 (light blue), HOXD12 (light gray) singly resulted in cell populations that trended lower. This decrease was reversed and significantly different upon combined transfection of Nacα siRNAs with Hox genes, compared to single siRNA transfections. C,D, Nacα KD alone significantly decreased the proportion of cardiomyocytes (ACTN1+) compared to controls, while treatment with HOXC12 or HOXD12 siRNA individually, had no effect. Combined transfection of Nacα and Hox genes reversed the decrease in cardiomyocyte population and was significantly different compared to Nacα KD alone and no longer different compared to controls. Gata4/6,MyoCD KD also significantly lowered the proportion of cardiomyocyte populations. E,F, Nacα KD increased the proportion of fibroblasts (TAGLN+) compared to controls. Combined KD of Nacα with any of the Hox genes did not alter fibroblasts numbers compared to controls but were significantly reduced compared to Nacα KD alone. G, Images of merged staining of cardiomyocyte, fibroblast, and endothelial cells shows the decrease in cardiomyocyte (red) and increase in fibroblast (green) staining when NACA is knocked down compared to controls. This is reversed upon co-KD with HOXC12 and HOXD12. Significance * vs. control. ^ vs. Nacα. # comparison is indicated by line. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.