Figure 5

Protein Kinase C activation reduces the formation of Insulin producing cells in organotypic pancreatoids. (a) Brightfield images of organotypic pancreatoids at day 1, day 2, and day 3 of culture, in either organogenesis media (control), or organogenesis media supplemented with 160 nm of PMA. Scale bar = 50 um. (b) Whole mount three-dimensional reconstruction of organotypic pancreatoid at day 9 stained by immunofluorescence, with endocrine beta cells marked by Ins (in green) and nuclei marked by Dapi (in blue). Scale bar = 50 um. (c) Quantification of the percentage of Insulin+ cell number compared to total nuclei number (Dapi+) from immunofluorescent stainings. Only Insulin+ cells with clear Dapi+ were included in the analysis, with peripheral staining in the control excluded. Error bars are SEM. N = 3. P < 0.05. (d) Quantitative PCR of Ins1 and Ins2 in control, PMA, and ILV treated pancreatoids at day3. Expression is normalized to Gapdh. N = 3 control, N = 5 PMA, and N = 3 ILV. Data are represented as mean+ SEM (*p < 0.05, **p < 01, ***p < 0.005 by unpaired Student’s t test).