Figure 1. The state of the cytoskeleton controls expression of the transcription factors NEUROG3 and NKX6–1 in pancreatic progenitors.

(a) Schematic of the differentiation protocol⁵ used for suspension differentiations and plate down studies. (b) Images of clusters at the beginning of stage 4 dispersed and plated onto ECM-coated TCP for the remainder of the protocol. Scale bar = 100 μm. (c) qRT-PCR of pancreatic genes at the end of stage 4 of cells plated on collagen I at the beginning of stage 4 compared to regular suspension clusters or clusters reaggregated after dispersion (Tukey’s HSD test, n = 4). (d) qRT-PCR of pancreatic genes at the end of stage 4 of cells plated on collagen I gels of varying heights at the beginning of stage 4. Increasing the height of collagen I gels fixed to TCP correlates with decreasing the effective stiffness experienced by cells (ANOVA, n = 4). (e) qRT-PCR of plated stage 4 cells treated with some commonly used cytoskeletal-modulating compounds to identify latrunculin A as a potent endocrine inducer (Dunnett’s multiple comparisons test, n = 4). (f) Immunostaining of plated cells at the end of stage 4 demonstrating that a 1 μM latrunculin A treatment increased NEUROG3+ cells and decreased NKX6–1+ cells when administered during stage 4. Scale bar = 50 μm. (g) Latrunculin A dose response of pancreatic gene expression added during stage 4 measured with qRT-PCR (ANOVA, n = 4). (h) Immunostaining of plated stage 4 cells treated for 24 hours with 1 μM latrunculin A, demonstrating depolymerization of F-actin but maintenance of PDX1 expression. (i) Western blot quantification of the G/F actin ratio within cells under different culture formats and treated with latrunculin A (n = 3). All data was generated with HUES8. All data is represented as the mean, and all error bars represent SEM. Individual data points are shown for all bar graphs. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.