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Figure S1. Efficient transfection of Anolis limb micromasses with the CMV promoter. (A) GFP positive cells are clearly visible in limb micromasses 48 hr after nucleofection. (B) Brightfield image of a subsample of dispersed limb micromass cells compared to (C) GFP fluorescence image of same cells reveals high efficiency of transfection.

Figure S2. Anolis hindlimb cells lose hindlimb-specific gene expression rapidly in culture. After the first passage of primary cell culture, the expression of both pitx1 and tbx4 is greatly decreased in Anolis hindlimb cells as measured by quantitative RT-PCR.

Figure S3. The expression of hoxc11 correlates with pitx1 expression in transfected Anolis forelimb micromasses and hindlimb controls. (A) Higher levels of ectopic pitx1 expression correlate with increased hoxc11 expression in transfected forelimb cells as measured by quantitative RT-PCR. (B) Non-nucleofected hindlimb micromass controls show a similar relationship between levels of pitx1 and hoxc11 expression.

Table S1. ΔCt values of assayed genes for pitx1-nucleofected treatment micromasses and GFP-nucleofected control micromasses and associated P-values for tests of significant differences of mean ΔCt values between treatments and controls.

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