Cell confinement and ciliogenesis. The experimental procedure for all panels is described in Fig. 2 C. (A) Cells on various micropattern sizes were serum starved for 48 h, fixed, and stained for F-actin (top) and acetylated tubulin (bottom with an inset magnification) to reveal the primary cilium. Two examples of cells on 500, 1,500, and 3,500 µm2 are shown to illustrate that most cells on 500 µm2 had a primary cilium, half had one on 1,500 µm2, and most cells on 3, 500 µm2 had no primary cilium. Bars, 20 µm. Inset bar, 4 µm. (B) The proportion of individual ciliated cells on 10 different micropattern sizes (n > 100 for each) is shown with the black curve. The proportion of ciliated cells in cell doublets (two cells per pattern) on 10 different micropattern sizes (n > 100 for each) is shown with the gray curve with respect to individual cell size. (C) Quantification of primary cilium length depending on micropattern size. Primary cilium length was shorter in larger micropatterned cells. (D) Quantification of ciliated cell frequency in cells plated on small (750 µm2) and large (3,000 µm2) micropatterns over time (n > 170 for each). The rate slightly increased from 24 to 48 h and then reached a plateau. (E) Quantification of primary cilium length on small (750 µm2) and large (3,000 µm2) micropatterns 72 h after serum starvation. Primary cilium length was similar to the one measured 24 h after serum starvation (compare with C). *, P < 0.05 and ***, P < 0.001. Plotted bars represent standard deviations. Horizontal bars represent mean values.
