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. 2016 Jun 23;6:28283. doi: 10.1038/srep28283

Figure 6. Requirements of cell signalling molecules for cholangiocytic cyst formation.

Figure 6

(a,b) Cysts cultured in the absence of suitable cytokines and reagents. ‘-EGF media’ means EGF was excluded but the other 5 reagents (HGF, Wnt3a, R-spondin-1, Y27632, and A8301) were included. EGF, R-spondin-1, and A83-01 were strictly required for the formation of cholangiocytic cysts. (a) Representative images of cysts. Scale bars, 100 μm. (b) For quantitation, cells were seeded into each 3D gel and cultured for 12 days in suitable culture media. The number of cells seeded in 3D gels was slightly different between sets, therefore we set the control as 1.0 and the relative number was calculated. Results are presented as mean ± SEM. Statistical analysis was performed with Student’s t-test (n = 9). (c) The number of cysts after 3D gel culture with various inhibitors. During 3D gel culture, p38 signal inhibitor (SB203580, SB202190), PI3K inhibitor (LY294002), and MAPK inhibitor (PD325901) were added. The addition of inhibitors started from 3 day of culture. The results are presented as the mean ± SEM. Statistical analysis were performed with Student’s t-test (n = 5).