Fig. 2

RA and FGF10 facilitated the differentiation of mESCs to SG placodes. A qPCR analysis revealed that salivary gland progenitor markers were upregulated after 2-days of RA treatment. The Ct values were compared to aggregates treatment without RA (control: CTRL). B The bright-light images of the aggregates in each culture condition (in the presence and absence of RA from day 4 to day 6) are shown. The aggregates cultured without RA failed to generate healthy SG placodes from day 7 to day 9. Scale bars: 200 μm. C The ratio of outer translucent salivary gland epithelium area became thicker after RA treatment, which became thinner in the control group (CTRL: aggregates cultured without RA). The area of cell death and outer epithelium were measured using ImageJ software. n > 5 independent experiments. D qPCR results showed that up-regulation of Sox9, but not Krt5 and Krt19, continued to day 9 in RA -stimulated aggregates. The Ct values were compared to aggregates treatment without RA (control: CTRL). E qPCR gene expression results after FGF10 treatment for 4 days, showed that salivary gland progenitor markers including Krt5, Krt14, Krt19, Sox9, and Ascl3 were upregulated. The Ct values were compared to aggregates treatment without FGF10 (control: CTRL). All qPCR results presented as the fold change compared with the mean ± S.D. and were normalized to GAPDH in three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by unpaired, two-tailed Student’s t-test