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Fig. S2 Quantification confirms reduced Fn deposition basal to the myocardium in embryos overexpressing hand2. (A) Representative transverse confocal section in an embryo expressing Tg(myl7:egfp) (green) exemplifies the method for quantification of Fn immunofluorescence (blue). Scale bar: 10 μm. For each image, we used ImageJ software to select a cardiac region of interest (r.o.i.) (solid rectangle) basal to a group of at least three cardiomyocytes and then measured the mean pixel intensity of the r.o.i. in the blue channel. Similarly, we measured the mean pixel intensity in a comparably sized r.o.i. (dashed rectangle) along the exterior cortex of the embryo. Measurements were taken from three consecutive confocal images per cryosection in three different cryosections per embryo, and at least three embryos were sampled for each condition. (B) Bar chart comparing the levels of Fn deposition in terms of mean pixel intensity (±s.e.m.) for each r.o.i. measured. Asterisk indicates statistically significant difference from wild type (P<0.0001, two-tailed unpaired Student′s t-test). In embryos overexpressing hand2, deposition of Fn adjacent to the cardiomyocytes is reduced <85%, whereas cortical deposition of Fn is relatively unaffected. (C) Bar chart comparing levels of Fn deposition adjacent to the cardiomyocytes, normalized relative to cortical Fn levels. Values represent the mean ratio (±s.e.m.) for each condition; asterisk indicates statistically significant difference from wild type (P<0.0001, two-tailed unpaired Student′s t-test). (D) Bar chart indicating levels of fn1 expression, relative to hprt expression, as detected by qRT-PCR in wild-type embryos and in embryos overexpressing hand2. Values represent the mean (±s.e.m.) from triplicate samples. fn1 expression is decreased 4-fold in embryos overexpressing hand2. PCR primers used were: fn1, 52-GAATCCCCGAGCCAATCAA-32 and 52-TTTTATGTTTCCCCGTTCCTT-32 hprt, 52-ATCCGCCTCAAGAGTTACCAA-32 and 52-TCTAGCAGCGTTTTCATCGTT-32.