Fig. 3

Colony, cell, and nuclear area of LSCs and relation of morphological parameters to p63 levels. A Fluorescence image of live LSC colonies (small cyan nuclei within cells very weakly stained with SiR-actin, magenta) growing within 3T3 feeder fibroblasts (much bigger cyan nuclei with larger nucleoli dots within nucleus, seen as intense cyan dots, within cells that are strongly stained for SiR-actin showing actin filaments, magenta). Cyan, NucBlue-Hoesht-33342; magenta, SiR-actin. Maximal projection of multiple z-stacks. B Area of colonies of LSCs, black dots, mean; black lines, standard deviations. C Nuclear area of individual LSCs within colonies, D Cell area of individual LSCs within colonies, E Nuclear area divided by cell area (nuclear to cytoplasm ratio, N/C) of each individual LSC within colonies. F Immunofluorescence image of fixed LSCs within the colony stained with SiR-actin (actin, magenta) and DAPI (DNA, cyan). Maximal projection of multiple z-stacks. G Nuclear area versus cell area across the population of LSCs within colonies. The black line represents the linear regression fit, or x = y line. H Immunofluorescence image of fixed LSCs within the colony stained with SiR-actin (actin, magenta), DAPI (DNA, cyan), and secondary antibody AlexaFluor488 conjugated to primary antibody raised against p63 4A4 (yellow). Maximal projection of multiple z-stacks. (left), p63 intensity in each cell versus its total area (middle), or its nuclear area (right). Black lines in (H) represent linear regression fit. I Immunofluorescence image of LSCs within the colony stained with secondary antibody AF647 conjugated to primary antibody raised against p63-α (dark blue), secondary antibody AF488 conjugated to primary antibody raised against p63 4A4 (magenta), DAPI (DNA, gray), and secondary antibody AF594 conjugated to primary antibody raised against tubulin (green). Maximal projection of multiple z-stacks. (left) p63-α versus p63 4A4 signal intensity in each cell; p63-α (middle) and p63 4A4 (right) intensity versus their nuclear area. Black lines represent linear regression fit