Fig. 4. Cells lacking PKP1 become more refractile in response to low Ca²⁺ concentrations compared to those expressing PKP1. Cells were seeded at high density and grown for 72 hours under normal Ca²⁺ concentrations (1.2 mM) before being subjected to a low Ca²⁺ switch and monitored using time-lapse imaging. (A) Phase contrast images at 10x magnification show confluent cell sheets after a low Ca²⁺ switch at time 0 (left panels), after 48 minutes (middle panels), and after 24 hours (right panels). Tight cell-cell contacts are lost in nullpB1 cells after 24 hours (top right panel), while areas of tight cell-cell contacts can be seen in nullPKP1 in addition to refractile cell edges (bottom right panel). Scale bar: 100 µm. (B,C) Digital images were analysed to determine the percentage of pixels above the mean background threshold at time 0. Graphs show the mean±s.e.m. Graph B compares cells derived from patient 1 (3-month-old male) with those derived from control 1 (31-year-old female), while graph C compares cells derived from patient 2 (5-year-old-male) with those derived from control 2 (45-year-old female) using separate time-lapse equipment (*P<0.05, ** P<0.01, ***P<0.005).