Fig. 3.

TRKB regulation of NGN3 in cultured human exocrine tissue. a Percentage of NGN3+ cells in human exocrine tissue cultures treated with Neurotrophic tyrosine kinase type 2 receptor (TRKB) agonist 7,8-dihydroxyflavone (78D, 0.1 μM), TRKB inhibitor ANA-12 (50 μM), protein kinase B (AKT) inhibitor API-1 (1 μM), combination of 78D and API-1, tyrosine kinase inhibitor CEP-701 (10 nM) or a combination of 78D and CEP-701 for 4 days. Mean ± SEM percentage of NGN3+ cells was determined using quantitative immunohistochemistry and normalized to dimethyl sulfoxide (DMSO) carrier control. Significance for ANA-12 determined by two-tailed homoscedastic Student’s t-test (n = 10 technical replica readings from 4 biological replicate exocrine cultures) and for other drugs by one-way ANOVA with Bonferroni post hoc testing (n = 10 technical replica readings from 3 biological replicate exocrine cultures), ***, p < 0.001. b-d Western blot analyses of signaling activity downstream of TRKB. b Levels of active protein kinase B (p(S473)AKT) following treatment with ANA-12 and 78D compared to DMSO carrier control. c Levels of active AKT following treatment with API-1 compared to DMSO carrier control. d Levels of active ERK1/2 (p(T202/Y204)ERK) following inhibition with FR180204 compared to DMSO carrier control. Protein loading level indicated by level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in b-e, total AKT in b-d and total ERK in e