Figure 4: ROCK1/2 inhibition reverses the effects of Slug in 3D acinar cultures.

(a) Primary acinar cells were isolated from wild-type B6 mice and seeded in 3D collagen. Trans-differentiation was induced with treatment with TGF-α (50 ng/ml) every 2 days for 5 days in the presence of the Rac inhibitor NSC23766 (20 μM) or the ROCK1/2 inhibitor Y27632 (10 μM). Formation of ductal structures was observed by phase microscopy and the number of ductal structures was quantified. (b) The primary acinar cells isolated from wild-type B6 mice were treated with TGF-α (50 ng/ml) every 2 days for 5 days and with the ROCK1/2 inhibitor Y27632 (10 μM) daily for 5 days. The mRNA samples were analyzed for ductal markers Pdx1, carbonic anhydrase II (CA II) and Sox9 expression after 1 day or 5 days of treatment, while the acinar marker Ptf1a expression was analyzed after 1 day and 3 days of treatment. The relative expression was normalized to the levels present on Day 1 of the TGF-α-only treated acinar cultures from wild-type mice. (c,d) Primary acinar cells were isolated from wild-type and Slug mice detailed in Fig. 1, seeded in 3D collagen, and trans-differentiation induced with treatment with TGF-α (50 ng/ml) every 2 days for 5 days in the presence of the ROCK1/2 inhibitor Y27632 (10 μM) or the ROCK1/2 inhibitor Fasudil (25 μM). Formation of ductal structures was observed by phase microscopy and the number of ductal structures was quantified. The results are representative of at least three independent experiments.