Figure 6. ROCK inhibition rescues the lumen phenotype and causes acinar expansion.

(A) Fluorescent images of representative acini formed by MDCK II cells induced for 7 d with doxycycline to induce mCherry-KASH1, and with ROCK inhibitor Y27632 (Y27) added on day 4 of culture (top panel, acinar development); images of acini formed during 7-d culture, followed by 2-d treatment with doxycycline to induce mCherry-KASH1 expression and Y27632 to inhibit ROCK (middle panel, acinar maintenance); and images of acini formed by uninduced MCF-10A cells followed by 2-d treatment with doxycycline to induce EGFP-SUN1L-KDEL expression and Y27632 to inhibit ROCK (bottom panel). The cartoon next to each panel indicates the period of three-dimensional culture and Y27632 treatment. Dx: doxycycline. (B) Quantification of data corresponding to (A) and untreated controls. DMSO served as vehicle control. At least 80 acini from at least three experiments were analyzed for the corresponding images in (A). Error bars represent ± SEM (* p<0.05, Student’s t-test with Bonferroni corrections for multiple comparisons). (C) Time-lapse fluorescent images of an acinus formed by MDCK II cells expressing mCherry-KASH1, which was treated with 40 μM Y27632 at 0 h (see Video S10). The size of the acinus increases and the thickness of the acinus decreases over a period of several hours after Y27632 treatment. Two nuclei (arrowheads) do not move significantly in the images for several hours after reaching the expanded state. (D) Plot shows time-dependent, pooled, averaged area and thickness of uninduced MDCK II acini (squares) normalized to the initial area and thickness respectively. Acini, pre-formed for 6 d, were induced with doxycycline for 24 hours to express mCherry-KASHΔPPPL (circles), or induced with doxycycline (Dx) for 24 h to express mCherry-KASH1 followed by 40 μM Y27632. The uninduced control is also shown. At least 5 acini from 3 experiments were analyzed, error bars are ± SEM.