Figure 5. Deregulated EMT in epicardium of PKR1^Wt1−/−.

(A) EMT markers (β-catenin, SNAI1 vim1 and myc) but not epicardial markers (Tbx18 and Wt1) were reduced in mutant hearts (n =  3, 5–6 individual hearts). (B) Representative western Blot analyses on whole heart extracts of mutant and control mice, utilizing β -catenin antibody and GAPDH as an internal control. Illustration shows β -catenin expression profile in the epicardium. Histogram shows that β -catenin protein levels were decreased in PKR1^Wt1−/− hearts (at least 3 times repeated, 5–6 mice per genotype, *p <  0.05). (C) PKR1 overexpression of epicardial cells significantly increased F-actin organization that was visualized by Phalloidin staining, indicating a mesenchymal-like cell formation. (D) Overexpression of PKR1 in embryonic epicardial cells by Adv-PKR1 increased EMT marker levels (n =  3). Data are expressed as mean ±  SEM (*p <  0.05). (E) Phalloidin staining in PKR1 deficient cells shows octagonal cell shapes without actin fibers after PK2 treatment. Representative illustration of ZO-1 internalization in epicardial cells 1h after PK2 treatment. (F) Explant cultures derived from control or PKR1^G5−/− embryonic hearts exposed to wound and healing process in the presence of PK2. PI3K/Akt inhibitor LY294002 reduced PK2-mediated migration. Quantification of migrating cells is shown in the histogram. Data are expressed as mean ±  SEM (*p <  0.05).