Figure 1.

Knockout of TMEM88 Decreases Cardiomyocyte Differentiation In Vitro, which Is Rescued by an Extended Treatment with an Exogenous Wnt Inhibitor

(A) Schematics of guide RNA (gRNA)-binding site (red box) on the first exon of TMEM88. Genomic sequencing of the TMEM88 KO clone 22 showed compound heterozygous alleles of 5-bp deletion and 7-bp deletion, shown below the wild-type (WT) sequence. The red boxed area is the gRNA target. The PDZ-binding domain motif, Val-Trp-Val, is located at the C terminus followed by a stop codon.

(B) Schematic diagram of monolayer-directed differentiation of cardiomyocytes. See Methods for details.

(C) Western blot data show the absence of TMEM88 in TMEM88 KO cells in day 5 differentiating cardiomyocyte progenitor cells. WT embryonic stem cell (ESC) was used as a negative control.

(D) Percentage of cardiac troponin T-positive (cTnT+) cells by flow cytometry at day 14 of differentiation. XAV939 was added from day 3 to 4 for the samples labeled “XAV1d.” XAV939 was added from day 3 to 5 for the samples labeled “XAV2d.” Graphs show mean ± SEM. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

(E) Expression of Brachyury, MIXL1, and EOMES in WT versus TMEM88 KO samples by qRT-PCR during cardiac differentiation. The results are displayed as normalized to WT ESC control or day1 control. Transcript levels in paired samples were not statistically different.