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The negative feedback loop does not function properly in the in vitro system, probably due to the insufficient induction of RIPPLY2.

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posted on 2022-01-13, 18:45 authored by Hajime Okada, Yumiko Saga

A Immunostaining of TBX6 in the in vitro PSM of iTbx6;iNICD (control), iTbx6;iNICD;Mesp2-CDS-KO, iTbx6;iNICD;Mesp2-Exon1-KO, and iTbx6;iNICD;Mesp2-Enhancer-KO upon Dox administration. Scale bars: 100 μm. B qPCR analysis of Tbx6 in the in vitro PSM (n = 6 cultures for each genotype). The expression level is presented as the ratio against iTbx6;iNICD line (control). P-values were calculated by the Mann-Whitney U test comparing iTbx6;iNICD (control) and iTbx6;iNICD;Mesp2-KO lines. Data are presented as the mean ± SD. Asterisk indicates significant (p < 0.05). C Schematic illustration of the generation of iMesp2 line and iRipply2 lines. D, E qPCR analysis of Mesp2 (D) and Ripply2 (E) in the in vitro PSM (n = 6 cultures for each genotype). The expression level is presented as the ratio against iTbx6;iNICD line (control). F Immunostaining of TBX6 in the in vitro PSM of iTbx6;iNICD (control) (left), iTbx6;iNICD;iMesp2 (middle), and iTbx6;iNICD;iRipply2 (right) upon Dox administration. Scale bars: 100 μm. G Western blotting analysis to monitor the degradation of TBX6 in the in vitro PSM of iTbx6;iNICD (control), iTbx6;iNICD;iMesp2, and iTbx6;iNICD;iRipply2 upon Dox administration. ß-Actin was used as the internal control of the protein lysate.

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