Figure 1: Rtfc is involved in thyroid differentiation of mouse ESCs.

(a) The design of TALENs to knock out the Rtfc gene. Boxes represent exons of the Rtfc gene. The coding regions are shown in black. (b) Sequences at the TALENs targeting site in two Rtfc knockout (KO) clones. (c) Knockout of Rtfc reduces the thyroid differentiation efficiency of mouse ESCs. WT ESCs and two Rtfc KO ESC lines were differentiated toward the thyroid lineage. The expression levels of thyroid markers were measured in day 7 differentiated cells (D7) by quantitative RT-PCR. Day 0 undifferentiated WT ESCs (WT, D0) were included as a control to show the activation of thyroid markers after differentiation. Data are shown as mean ± SD (n = 3). (d) Western blot to detect the overexpression of FLAG tagged GFP, WT and V205M RTFC proteins in mouse ESCs. Stable mouse ESC lines overexpressing FLAG tagged GFP, WT and V205M RTFC were established and subjected to Western blot. A triangle indicates the band of FLAG-GFP, and an asterisk marks the band of FLAG-RTFC. Cropped blots are shown, and full-length blots are presented in Supplementary Figure S5. (e) Overexpression of WT and V205M RTFC promote the thyroid differentiation of mouse ESCs. ESCs overexpressing GFP, FLAG tagged WT and V205M RTFC, were differentiated toward the thyroid lineage. The expression of thyroid markers in day 7 differentiated cells (D7), as well as in day 0 undifferentiated GFP ESCs (GFP, D0), were analyzed. Data are shown as mean ± SD (n = 6, including 2 independent clones and 3 technical replicates).