Fig. 6: Short-term WNT5A treatment activates the JNK/c-JUN pathway in human EPs.

a hESC-derived EPs stained for FZD3 and INSM1. Scale bar = 50 µm. N = 3 independent experiments. b Blocking FZD3 receptor in EPs leads to ~2.5-fold decrease in INS+ cells compared to untreated control at day 3. Mean ± SEM and a p value (unpaired two-tailed Student’s t test) are shown. N = 3 independent experiments. c Scheme to investigate by RNA-sequencing global gene expression changes in human EPs induced by short- (12 h) and long-term (5 days) WNT5A treatment. d WNT5A treatment shifts the transcriptional profile of hESC-derived EPs toward that of β-cells. Z-score of normalized RNA-sequencing expression values of selected genes with at least one pairwise difference of q < 0.05. e GSEA plot from RNA-sequencing showing short-term EP treatment with WNT5A activates JUN pathway. f GSEA heatmap showing JNK target upregulated genes in hESC-derived EPs after short-term WNT5A treatment. g Predicted significantly upregulated TFs in EPs after short-term WNT5A treatment, showing high c-JUN upregulation as analyzed by TFactS. h Western blot for total and phosphorylated JNK after WNT5A treatment of EPs. Beta-actin is a loading control. Multiple bands of p-JNK and JNK correspond to 54 and 46 kDa isoforms. N = 3 independent experiments. i Phospho-c-JUN and INSstaining in EPs treated for 24 h with WNT5A or in untreated (UT) control. DAPI marks nuclei. Scale bar = 100 µm. N = 6 independent experiments. j Quantification of p-c-JUN+ cells shown are a percentage out of total (DAPI+) cells in untreated (UT), and WNT5A-treated EPs showing a 6-fold increase in the number of c-JUN+ cells. Mean ± 95% CI and a p value (unpaired two-tailed t-test) are shown. N = 6 independent experiments. k JNK inhibition by SP600125 lowers INS+ cell induction. EPs were treated for 3 days with 20 µM or 40 µM SP600125, and INS+ were quantified and presented as fold change compared to vehicle (DMSO) treated control. Co-treatment of EPs with SP600125 and WNT5A (blue) does not rescue INS+ cell induction. Mean ± 95% CI and p values (Tukey’s multiple comparison test vs. control) are shown. N = 5 independent experiments.