A: Representative in vitro images of nuclear GATA6+nuclear GLI3R (upper), nuclear GATA6+cytosolic GLI3R (middle) and cytosolic GATA6+cytosolic GLI3R (bottom). B: Quantitation of subcellular localization of GATA6 and GLI3R. N<C: predominantly cytoplasmic, N = C: similarly in cytoplasm and in nucleus, N>C: predominantly nuclear localized. GATA6 mutants, indicated at the bottom, are shown in Fig 5E. The number of cells examined for each set of transfection is indicated in the panel. C-H: Representative images of the anterior-proximal mesenchyme of hindlimb buds at E10.25. C, E, G: wild type, D, F, H: Gata6 cKO. I: Quantitation of subcellular localization of GLI3R in the anterior-proximal mesenchyme of hindlimb buds at E10.25. Gray and black bars represent wild-type and Gata6 cKO samples, respectively. The graph shows percentage of GLI3R localization patterns, such as predominantly nuclear (N>C), similarly in the nucleus and cytoplasm (N = C), or predominantly cytoplasmic (N<C). A total of 597 cells from three wild-type embryos and a total of 528 cells from three Gata6 cKO embryos were examined. * indicates P<0.05. J: Western blot of nuclear fractions from anterior part of wild-type and Gata6 cKO hindlimb buds at E10.25–10.5. Histone H3 (H3) is included as a loading control.