Figure 1. Genomic and proteomic analysis identifies protein dephosphorylation events that correlate with commitment.

(a) Schematic of experimental design. (b) Colony formation by cells harvested from suspension at different times. Representative dishes are shown together with % colony formation (n = 2 independent experiments, n = 3 dishes per condition per experiment; p-values calculated by Tukey’s multiple comparison test). (c) Cells isolated from suspension at different time points were labelled with anti-involucrin (IVL) antibody (green) and DAPI as nuclear counterstain (blue). IVL-positive cells were counted using ImageJ (n = 3 independent cultures; more than 300 cells counted per condition. p-value calculated by two-tailed t-test). Scale bars: 50 μm (d) RT-qPCR quantification of ITGα6, TP63, IVL and TGM1 mRNA levels (relative to 18 s expression) (n = 3 independent cultures). (e) t-SNE plot of genome-wide transcript expression by keratinocytes placed in suspension for different times. The t-SNE algorithm takes a set of points in a high-dimensional space and finds a faithful representation of those points in a lower-dimensional space, typically the 2D plane. (f) Heatmap representing hierarchical clustering of differentially expressed proteins (p<0.05). (g–i) Dot plots correlating expression of significantly differentially expressed peptides (p<0.05) that change twofold relative to 0 hr in at least one of the time points, with their corresponding differentially expressed transcripts. Pearson correlations (r) are indicated. (j) Histogram of normalised SILAC ratios corresponding to high confidence phosphorylation sites that differ between 0 and 4 hr. (k) Scatter plot correlating log₂ normalised SILAC ratios for total protein changes (y-axis) with log₂ phospho-peptide ratios (x-axis) between 0 and 4 hr. (b, c) *p<0.05; **p<0.01; ns = non-significant).

Figure 1—figure supplement 1. Clonal growth, genomic and proteomic analysis of suspension-induced terminal differentiation.

(a) Keratinocytes harvested after 0, 1, 2, 4, 6, 8, 10, 12 or 24 hr in suspension in methylcellulose were seeded at 100, 500 or 1000 cells in 10 cm² culture dish on mitotically inactivated J2 3T3 feeders and cultured for 12 days. Following staining, colonies were counted using ImageJ (n = 2 independent experiments with three replicate dishes per experiment; p-value calculated by two tailed t-test). (b) RT-qPCR quantification of IVL and TGM1 mRNA levels at different times in suspension (n = 3 independent cultures; p-value calculated by two tailedt-test). (c) RT-qPCR quantification of DLL1, Ki67 and Lrig1 mRNA levels at different times in suspension (n = 3 independent cultures; p-value calculated by one-way ANOVA). (d) Hierarchical clustering of significantly expressed transcripts at 0, 4, 8 and 12 hr in suspension (each time point represents the mean value of n = 3 independent experiments). (e) GO analysis of differentially expressed genes upregulated at 4, 8 and 12 hr relative to 0 hr. The bar plots represent –log₁₀ of p-values of the identified GO terms. (f) Schematic of SILAC-Mass Spectrometry labelling protocol. (g) GO analysis of proteins ranked in the order of their expression level (fold increase relative to 0 hr) at 4, 8 and 12 hr. GO terms were fetched for individual proteins rather than for clusters of proteins. Bar plots represent –log₁₀ of the p-values of the identified GO terms. (a–c) Error bars represent s.d. *p<0.05; **p<0.01; ***p<0.005; ns = non significant.