Figure 6. Low cyclin D causes cell-cycle arrest in small cells.

(A) Cell membranes of Tet-On p27 and Tet-On p27ck MDCK cells in ME + 4 days; dox added at $\text{t}=0$ h.

(B) RNA sequencing data from monolayers prepared in (A). Data are averaged transcripts per million from 3 experimental replicates. Inset: zoom in of genes in indicated box, cyclin D genes expression levels are highlighted in red.

(C) DNA staining and anti-cyclin D1 (cyD1) immunofluorescence staining in MDCK monolayers at ME + 3 days. Dox is added at either $\text{t}=0$ (+dox p27) or at $\text{t}=\text{ME}+2$ days (delay +dox p27).

(D) Quantification of anti-cyD1 intensity from immunostaining data as a function of nuclear area. Intensity is normalized in each experiment to a maximum value of 1. P27/p27ck are monolayers with a mixture of Tet-On p27 and Tet-On p27ck cells. $\left({\text{N}}_{\text{+dox}}=4,564[3],{\text{N}}_{\text{Delay+dox}}=7,515[2],{\text{N}}_{\text{p}27/\text{p}27\text{ck}}=11,080[4]\right)$.

(E) Labeled cell membranes in Tet-On cyclin D1 T286A T288A (CyDAA) MDCK monolayers at ME + 3 days without dox (−dox) or with dox added at $\text{t}=0$ (+dox CyDAA).

(F) Cell volume measured in resuspended Tet-On cyclin D1, Tet-On cyclin D T286A T288A, and Tet-On 12sE1a cells at ME + 3 days without dox (−dox) or with dox added at $\text{t}=0$ (+dox CyD, +dox CyDAA, +dox E1a). $({\text{n}}_{\text{−dox}}=13,513[\text{N}=10],{\text{n}}_{\text{CyD}}=7,240[\text{N}=2],{\text{n}}_{\text{CydAA}}=9,092[\text{N}=3],{\text{n}}_{\text{E}1\text{a}}=6,457[\text{N}=4])$ (*p < 0.05, **p < 0.01 from comparison of experimental means). See also Figures S9 and S10.