FIGURE 5.

Impaired cardiac differentiation in inhibitors of DNA-binding proteins (ID) double-knockout (KO) human embryonic stem cell (hESC) lines. a) Immunoblotting analysis of ID1 and ID3 in three IDs KO hESC lines (ID KO-#1/#2/#3) and three wild-type (WT-#1/#2/#3) cell lines. α-TUBULIN was used as a loading control. The experiment was performed three times for each cell line; n=3. b) Fluorescence-activated cell sorting (FACS) analyses (left) and quantification (right) of the percentages of cTnT⁺ cells on day 16 of cardiomyocyte (CM) differentiation. n=3, three repeats for each cell line; n=3, three cell lines in each group. c) Immunostaining of α-Actinin (green) and cTnT (red) in cells differentiated from WT-#1 and ID KO-#1 cell lines. 4′,6-diamidino-2-phenylindole (DAPI) was used as a counterstain and is shown in blue. Scale bars=50 μm. d) Immunoblotting of EOMES/ID1/ID3 and α-TUBULIN (as a loading control) at different stages of CM differentiation in WT and IDs KO cell lines. e) The mRNA levels of Brachyury (T), MIXL1, TBX5, GATA4 and ISL1 during CM differentiation were measured by quantitative PCR. n=3, three technical replicates for one cell line. All results are presented as mean±sem. **: p<0.01, ***: p<0.001, ****: p<0.0001 versus WT and IDs KO as determined using b) unpaired t-tests and e) two-way ANOVA with post hoc tests (Sidak's multiple comparisons test).