Transcriptome profiling of the keratinocyte differentiation process identifies dynamic expression profiles

Differentiation was initiated in human primary keratinocyte stem cells by the addition of CaCl₂ [3, 18]. Cells were harvested over 45 hours at 5-hour intervals with three biological replicates. At each time point cells were lysed, mRNA was isolated, and expression profiles were measured using Agilent microarrays [3] (Fig 1A and S1 Table). 21,113 probes mapping to 16,720 genes were detected as expressed. We defined genes that are changing (‘dynamic’) and those that are constantly expressed (‘non-dynamic’) using a 2-fold expression change cut-off and a Chi-squared test on time points of biological replicates (see methods). As a more stringent criterion for classifying dynamic genes, we used a threshold of 4-fold, defining these genes as ‘super-dynamic’ (S1 Table). In summary, out of the 16,720 expressed genes, 6,137 are ‘non-dynamic’ (P> = 0.01, Chi-squared test, and fold change <2), 6,096 are ‘dynamic’ (P<0.01 and fold change > = 2), 1,317 of the dynamic genes are also ‘super-dynamic’, (P<0.01 and fold change > = 4), and 4,487 genes do not fall into one of the above categories (classified as ‘unresolved’). In general, we used the super-dynamic genes for our analyses, unless otherwise stated.

Classification of proteins encoded by the super-dynamic genes based on DAVID [20,21], UniProt [22], and manual literature searches (e.g. [23]) (S1 Fig) uncovered a large fraction of proteins involved in: i) signaling (transcription factors, and the adhesion, chemokine, calcium, immune/Toll, ubiquitin-like, apoptosis, Wnt, TGFβ, interferon, and Notch signaling pathways (S2 Fig) (32%); ii) housekeeping functions (cytoskeleton, cell cycle, solute transport carriers, histone, and chaperone proteins, or formation of tight junctions (S3 Fig) (22%); iii) metabolic enzymes for lipid, amino acid, steroid, purine, and flavine interconversions and lipid binding (S4 Fig) (13%), iv) proteins needed for the progressive steps towards the formation of the cornified envelope (metallo and serine proteases), crosslinking enzymes (e.g. transglutaminases) and substrates (e.g. loricrin, involucrin, and small proline rich proteins) that provide structural stability and elasticity, keratins (mechanical resistance), and lipid modifying enzymes (water repellence) (S5 Fig) (8%) [23]. For 8% of the super-dynamic genes only one Pfam domain prediction can be assigned, while 17% of them have no known function or Pfam domain annotations. Thus, the process of keratinocyte differentiation is accompanied by concerted changes in metabolic, signaling, and housekeeping pathways. We also confirmed the induction of known markers for keratinocyte differentiation (involucrin [IVL], filagrin [FLG], cystatin [CSTA, CSTB]) (S1 Table) [23]. Involucrin expression was additionally confirmed on the protein level by immunostaining (S6 Fig).

Unsupervised clustering partitions the temporal profiles of super-dynamic genes into eight clusters

We used K-means clustering to classify the temporal profiles of the 1,317 super-dynamic genes and identified eight optimized clusters (Fig 1B). The clusters are arranged with opposing temporal profiles on the top and on the bottom: clusters 1 and 5 contain early highly transient expressed/repressed genes, clusters 2 and 6 contain early transient expressed/repressed genes, clusters 3 and 7 contain early highly sustained expressed/repressed genes, and clusters 4 and 8 contain early sustained expressed/repressed genes. Consistent with the function of the Ets transcription factor ELF3 in promoting differentiation of keratinocytes [24,25], we see strong immediate and sustained expression of ELF3 (cluster 3), followed by delayed expression of genes regulated by ELF3 like KRT4 and TGFβ (cluster 8), and the envelope protein SPRR2A (cluster 3) [26,27].

The clusters reveal a moderate stage-specific expression of functionally related protein classes and biological processes. For instance, proteins related to the formation of the cornified envelope are strongly induced at later time points (clusters 3, 4, 5, and 8). Cluster 7 (repressed genes) is enriched for cell cycle-related proteins in the housekeeping category, as expected during the onset of differentiation [28]. This cluster has an antagonistic behavior when compared to the cell-cycle enriched cluster published in a cellular reprogramming study [4]. Signaling-related pathways in clusters 1 and 2 involve transiently expressed transcription factors, cytokines, and proteins involved in Wnt, adhesion, TGFβ, and TNF signaling (S1 Table). Likewise, enzymes involved in lipid and amino acid metabolism are present in most clusters. The absence of a strong association between functional categories and clusters suggests a functional replacement within sub-categories, possibility brought about in some cases by dynamic rearrangements within protein complexes.

During keratinocyte differentiation, a switch in the expression of paralogous gap junction subunits is needed for the changes in gap junction permeability required for epidermis formation [29], which we see in our study (Fig 2A). To see if this is a general case for all paralogous pairs, we annotated expressed genes in keratinocytes with homology information from EnsemblCompara [30] limiting our analysis to paralogous pairs in which both genes are dynamically expressed, thereby reducing our set from 11,582 to 2,260 paralogous pairs. Computing Pearson correlation metric for all pairs, we found 950 to have highly correlated (r> = 0.6, P<0.07) dynamic expression profiles during skin differentiation with a subset of 235 qualifying as super-dynamic, and 281 pairs to have highly anti-correlated dynamic expression (r< = -0.6, P<0.07) with 19 of these pairs being super-dynamic (Fig 2B and S2 Table). When compared to super-dynamic and dynamic random pairs, we found that paralagous pairs are more likely to have correlated expression (Wilcoxon rank sum test; P = 4.5e-37, for super-dynamic S7A Fig; and P = 5.4e-61 for dynamic, S7B Fig). An example where gene duplicates can act together to bring about a functional change is the formation of cornified envelope (e.g. the transglutaminase substrates, small proline-rich proteins) (Fig 2C and S8 Fig). This could suggest a requirement for increased gene dosage in particular specific stages of cell differentiation as the likely reason for the duplication of these genes [31–33].

We also found nineteen anti-correlated pairs where both proteins are super-dynamic (Figs 2D–2F and S9 Fig). Examples include PLEKHA 6/7/4 (Fig 2D; for PLEKHA-7 a functional role has been demonstrated in recruiting paracingulin to tight junctions [34]), WNT7A/7B/5A (Fig 2E), NDRG1/2/4 (Fig 2F; NDRG2 is expressed in response to TGFβ and inhibits proliferation [35]) and CCNA/B proteins (S9 Fig). One explanation for this observation might be that there is temporal sub-functionalization, with one paralog being expressed early, the other late during the course of differentiation as the paralogs have taken on different functions. Therefore, the expectation would be that anti-correlated paralogous pairs are more divergent in sequence level than correlated ones. Using Pfam domain annotations we compared correlated to anti-correlated dynamic paralogous pairs (here we used dynamic pairs to increase the numbers for statistical analysis since not every protein has Pfam annotations) and found that correlated paralogous pairs are more similar in their Pfam domain composition (different domain, or domain missing) than anti-correlated pairs (1.3 versus 1.7; Wilcoxon rank sum test; P = 0.009) (Fig 2G and S10 Fig). Likewise, anti-correlated dynamic paralogous pairs have greater sequence divergence (S11A and S11B Fig) and a greater number of differences in amino acid sequence length (S11C and S11D Fig). Furthermore, anti-correlated genes have a higher fraction of evolutionary old duplicated genes compared to correlated genes (S12 Fig).

In summary, dynamic paralogs are enriched in gene pairs that are correlated in expression. This suggests that a requirement for increased gene dosage is the likely reason for the duplication of these genes. Paralogous genes with anti-correlated expression changes have greater amino acid sequence and Pfam domain divergence. This implies that anti-correlated paralogs have taken on specialized roles during differentiation.

Expression changes reveal dynamically expressed genes encoding proteins in complex with proteins encoded by non-dynamically expressed genes

The finding that divergent paralogous pairs tend to have anti-correlated expression patterns could suggest replacement of subunits in protein complexes to expand functional complexity. To see if this is a general feature of protein complexes during differentiation, we obtained a list of protein complexes from the CORUM database (a resource of manually annotated protein complexes from mammalian organisms [36]), and mapped it onto the non-dynamically expressed, unresolved, or dynamically changing proteins (Fig 3A and S13 Fig and S3 Table). After removing complexes containing unresolved genes, 19% of the complexes did not have significantly changing gene expression for any of their subunits during the keratinocyte differentiation process (all non-dynamic), 26% of the complexes had dynamically changing expression for all subunits, and for half of the complexes (55%) we find a mix of behaviors (here called ‘di-chromatic’) (Fig 3B). We represented the previously clustered 1,317 super-dynamic genes in the context of complexes to which they belong and also assigned dynamic genes to each one of the eight super-dynamic clusters through comparative correlation analysis with average expression profile of the aforesaid clusters (Fig 4; S1 Network). We found that super-dynamic genes in the same complex or closely connected in the network often belong to different clusters (72%). However, this ratio decreases to 43% if clusters with similar behaviors (1 and 2, 3 and 4, 5 and 8, 6 and 7) are combined.

To further characterize the expression profiles of di-chromatic complexes, we classified the CORUM complexes into functional groups (S3 Table). Then we represented the expression changes for functionally related complexes or complexes sharing components (Fig 5 and S14–S16 Figs). Among many cellular processes known to be important for keratinocyte differentiation or general cell differentiation (Fig 5A), we find examples of complexes with a stable component or core and a dynamic periphery. Examples in cell signaling include EGFR and TNF-α/NFκB pathways. EGF signaling is important for keratinocyte differentiation [37] and we find two complexes involved in its recycling and degradation. In both complexes, one component (STAM2 and CBLB) is constant and the other is super-dynamic (RIN1 and SH3BKP1, respectively), changing in opposite directions in the two complexes. RIN1 regulates EGFR degradation in cooperation with STAM [38], and SH3BKP1 prevents epidermal growth factor receptor degradation by the interruption of c-Cbl-CIN85 complex [39]. Thus, the opposite behavior of these complexes will favor EGFR stabilization and consequently, EGF signaling and keratinocyte differentiation. In this respect we also find anti-correlated changes of the paralogous receptors EGFR and ErbB2, which slow down EGFR recycling through heterodimer formation [40].

The TNF-α/NF-κB signaling pathway is required for normal epidermal development and homeostasis [41–43]. As the CORUM database is not complete [44], we combined the CORUM complex information with a detailed structural and pathway analysis based on the literature [45, 46] (S15 Fig). We identified a core TNF receptors/scaffold complex (TNFRSF1, TRADD, RIPK1, TRAF2, IKBG1, CHUK, CDC37 and HSP90AA1) associated with a MAPK pathway (i.e. MAP2K7/ and MAPK8/9) which is composed of non-dynamic and unresolved genes, while the rest of the pathway in general is composed of dynamic components. This is an example of a stable core module associated with a dynamic peripheral module. Most of the super-dynamic genes are found mainly in the transiently expressed clusters 1 and 2, suggesting relevant concerted changes during keratinocyte differentiation (Fig 5B). MAP3K8 (Cot) is in cluster 4 (early activated and sustained expression) and can form larger complexes containing NFKB1, thereby promoting signaling. Likewise, CFLAR is in the same cluster 4 and is an inhibitor of FADD thereby preventing apoptosis. NF-κB/RelA has been shown to control cell-cycle exit in keratinocytes [47].

NF-κB-independent signaling commencing at the level of the non-dynamic core connects to MAPKs (such as NIK, NAK, TAK1, and Cot) and PKC isoforms [48]. Interestingly we see that TRAF1 expression is in the same cluster as the NF-κB components. It has been described that TRAF1 promoter has NF-κB binding sites and is strongly activated by TNF-α [49]. TRAF1 cannot bind to the TNF receptor directly, but is recruited though binding to TRAF2 [50] and overexpressing TRAF1 does not affect the interaction between TRAF2 and FADD [51], suggesting a compatible complex formation. TRAF1 binding to TRAF2 results in blocking apoptosis [52] and analysis of TRAF1-/- mice suggests that TRAF1 inhibits TNF-α signaling [49]. Thus, TNF-α/NF-κB induced dynamic expression of TRAF1 creates a negative feedback loop that could mediate anti-apoptotic functions [52] and decrease NF-κB activation without affecting other possible constitutive function of TNF receptor. We propose that the stable core module connects to both a dynamic peripheral module important for cell cycle arrest during differentiation (via NF-κB heterodimers and prevents apoptosis (via TRAF1), and several stable modules that function independent of NF-κB by binding to TRAF or the IKK complex and which should play a housekeeping function [48] (see S15 Fig for a summary of different signaling functions).

In the Notch/γ-secretase pathway [53], the γ-secretase (APH1B, promoting differentiation) is up-regulated and delta like 1 (DLL1, blocking differentiation) is down-regulated (Fig 5B). Both proteins associate to the same stable core (PSENEN and NCSTN), reinforcing a biological role for di-chromatic complexes. In the ubiquitination/degradation pathway, the CAND1 assembly factor of the E3 ubiquitin ligase complex [54] is up-regulated transiently and the S-phase kinase-associated protein 2 (SKP2, promoting cell cycle) is down-regulated as the target of the E3 ubiquitin ligase complex (S14A Fig). Skp2–Skp1 abrogates the inhibitory influence of CAND1 on the neddylation of Cul1 by promoting the dissociation of the cullin–CAND1 complex, whereas substrate, together with substrate-presenting components, prevents the action of CSN to deneddylate cullin [55]. It has been described that high levels of Skp2 are needed for proliferation in stratified epithelia [56]. In this respect it is noteworthy that CAND1 also causes elevation of p27, which has been demonstrated to be important during pre-adipocyte differentiation [57].

Finally, two complexes with interesting di-chromatic behavior are related to chromatin remodeling and known to be important for regulating gene expression changes during keratinocyte differentiation [58,59]. Emerin is a nuclear membrane protein, which is involved in tissue-specific gene regulation [60] and expressed constantly during the keratinocyte differentiation (S14C Fig). The emerin binding protein LMO7 is a cell type-specific transcription factor that is strongly up-regulated during differentiation. It acts by escaping actin (ACTB)-mediated inhibition [61,62] and therefore its levels need to increase strongly. At the same time, Laminin B1 (LMNB1; important for DNA replication) is transiently down-regulated. Finally, the BAF (SWI/SNF) complexes are known for their combinatorial assembly providing functional specificities [63,64] (S14D Fig). BRCA1 can directly interact with the BRG1 subunit of the SWI/SNF complex and is down-regulated during differentiation. This may liberate the SWI/SNF complexes to take part in chromatin remodeling, which are either constantly expressed or moderately down-regulated.

Generalizing, there are three different types of assembly motifs: In some complexes, dynamic genes are added or removed from the complexes and these are predicted to be compatible (AND; [14]) interactions (e.g. TNF-α/NFκB signaling and Notch complexes). For other complexes we observed opposing expression changes of subunits, which are potentially competing for the same binding interface (XOR interactions; i.e. CULIN SKP2 CAND1 [14]). Finally, there are large assemblies where we observed a mixed behavior (i.e. EGFR/TGF-β; S16 Fig). In summary, we find a di-chromatic behavior with a stable core for many complexes involved in cell differentiation. We suggest that the different assembly motifs with respect to compatible (AND) and mutually exclusive (XOR) surface interactions should be classified using 3D structural information to analyze which type of assembly motifs dominate during keratinocyte differentiation.

Structural analyses identify mutually exclusive surface interactions as enriched in dynamic genes

Proteins that bind mutually exclusively to the same domain on a shared binding partner protein prevent each other’s binding through steric hindrance depending on concentration and localization [10, 13, 14]. Steric hindrance and competition could be a mechanism to achieve cell type-specific functions if a competing protein is expressed at a higher level in a specific cell type or tissue [14]. To determine if the replacement of subunits of complexes during the differentiation process happens at mutually exclusive surface interactions, we structurally analyzed all CORUM complexes with less than 20 members using the SAPIN software framework (S17 Fig) [19]. SAPIN identifies the protein regions that could be involved in an interaction, provides template structures, and then performs structural superimpositions to identify compatible and mutually exclusive interactions. If a protein has at least two interacting partners, the domains mediating the interaction are superimposed on the reference domain, and the interacting domains are analyzed for compatibility (AND) or mutual exclusiveness (XOR) (S4 Table). Next, we combined expression classification during keratinocyte differentiation (non-dynamic vs. dynamic) with compatible and mutually exclusive interaction types (S18 Fig). Out of six possible cases (obviating the unresolved group), three cases were selected that we could interpret in terms of competition (Fig 6A). In case 1, the hub protein (common protein with at least two different binding partners) is non-dynamically expressed while the attachment proteins are dynamic. In case 2, all three interacting proteins are dynamic. In case 3 all three interacting proteins are non-dynamic. Interestingly, case 1 is significantly enriched for gene products with mutually exclusive surface interactions (XOR) (61% compared to 39%, Fisher’s exact test; P = 1.9e-6), reinforcing our hypothesis that dynamic genes tend to be involved in competing interactions for a common (constitutively non-dynamically expressed) binding partner (Fig 6 and S19–S21 Figs). Case 3 is significantly enriched for AND (61% compared to 39%, Fisher’s exact test; P = 2.03e-12), representing stable complexes that do not change their composition during the differentiation process. Case 2 (all three proteins dynamically changing) is also enriched for XOR interactions compared to AND (65% compared to 35%, Fisher’s exact test; P = 8.66e-04). When focusing on the 32 XOR nodes with at least two super-dynamic genes, 26 XOR nodes contain genes from at least 2 different clusters suggesting that opposing or at least different expression profiles may impose different functional outputs possibly to compete at single interface hubs. Thus, variation in the concentration of proteins (belonging to different clusters) that bind to XOR nodes may cause complex re-assembly through competition and thus achieve a different functional output in the differentiated keratinocytes or during the process of differentiation.

Microarrays, data normalization and filtering

The time-course microarray data analyzed in this work was generated in our previous study [3]. However, in this section of the methods, we re-outline the details of this experiment. Total RNA in the amount of 500 ng from calcium treated primary human keratinocytes collected every 5 hours in triplicates for a total of 45 hours (i.e. 10 samples) and labelled using Agilent LowInputQuick Amp Labelling kit following manufacturer instructions. mRNA was reverse transcribed in the presence of T7-oligo-dT primer to produce cDNA, The cDNA was then in vitro transcribed with T7 RNA polymerase in the presence of Cy3-CTP to produce labeled cRNA and this labeled cRNA was in turn hybridized to Agilent Human Gene Expression 4x44K v2 microarrays (ID026652). Signals from probes were obtained by the Agilent’s Feature Extraction custom software, were corrected for background noise using the normexp method [80] available in the R package limma from Bioconductor, and then normalized between arrays to assure comparability across samples using quantile normalization [81]. Lastly, the dataset was log2-transformed. The dataset was filtered to remove and collapse all Agilent control spots and to only include probes that were present above stochastic background expression. To do this we filtered any probes with expression profiles completely below 7 which is a figure obtained by taking the median of the entire dataset and rounding it down to a single digit. This procedure yielded 21,113 probes, which were then mapped to 16,720 genes where the probe with the highest mean expression represents a gene. For some analyses such as K-means clustering the dataset was mean-centered and scaled such that the mean of each gene is zero.

Classification into constitutive and dynamic genes

To distinguish genes that are changing (i.e. transiently or continuously up- or down-regulated) from those that are constant (i.e. stably expressed) across our differentiation time-course, we used Chi-squared test where for each gene, the expected value at each time point is equivalent to the gene’s average expression across all 10. The equation is as follow: where N is the number of samples or time points (i.e. 10), is the mean expression of gene x across three experimental repeats at time point i, and is the overall mean expression of gene x across all 10 time points and all repeats. Note that in this formula there is no normalization of by the experimental error computed as . Instead we computed a universal error and scaled the X² distributions of our data to the standard X² distribution for 9 degrees of freedom.

In addition to Chi-squared test we employed an empirically derived fold change threshold of 2 and a strict threshold of 4. Each gene’s expression peak was compared to its trough to ensure the difference satisfies the appropriate threshold and the genes were classified as follow:

1. non-dynamic genes have to satisfy P(X²)> = 0.01 and fold change < 2
2. unresolved genes either have P(X²)<0.01 and fold change < = 2 or P(X²) > = 0.01 and fold change > = 2
3. dynamic genes P(X²)<0.01 and fold change> = 2.
4. super-dynamic genes are a subset of dynamic genes which have fold change> = 4

We also conducted corrections for multiple testing using Benjamini’s method. While the above analysis had classified 6,096 genes as dynamic (1,317 as super-dynamic), after Benjamini correction for multiple testing and applying a p-value cutoff of 0.01 (1%), these two numbers remained the same while the number of non-dynamic genes increased from 6,137 to 6,413 and the number of unresolved genes decreased from 4,487 to 4,211. Thus, a subset of 200 genes got shifted from the unresolved category to the non-dynamic category after Benjamini correction. Since the majority of our analyses were based on dynamic and super-dynamic genes such as clustering and mapping to complexes and these classes did not change after multiple testing correction, we kept the gene classes as originally defined without Benjamini correction.

Statistical and bioinformatics analyses

We used R version 2.14 for the majority of the statistical analyses along with Perl and AWK for text processing. K-means clustering was performed using the R kmeans function. To optimize and select the K value or the number of cluster centers, we exhaustively performed K-means clustering with K value ranging from 2 to 500 and then for each set of clusters, computed an F-score as well as an average silhouette score available from the standard statistics and cluster packages, respectively. The K value, which resulted in the highest average silhouette score and F-score, was selected as the optimal number of cluster centers.

Protein complex analysis

A reference set of complexes was obtained from the CORUM database [36] of curated mammalian protein complexes. Out of a total of 1,331 human complexes available in the latest release (February 17^th, 2012), 44 homo-oligomer complexes consisting of only one type of protein were filtered out. 84 complexes were removed, which were fully non-expressed across our skin differentiation expression time course. 154 (<13%) complexes contain subunits that are partially non-expressed in keratinocytes while 1,049 complexes contain subunits, which are all expressed during skin differentiation. The resulting set of 1,203 complexes consists of 2 or more distinct proteins per complex.

Paralogous gene annotations

Paralog annotations were obtained from EnsemblCompara through the BioMart portal (database version EnsemblGenes71). The method identifies true paralogs by computing a phylogenetic tree across the whole set of protein-coding genes with one pipeline which includes TreeBeST [33].

Structural analysis

Analysis of mutually exclusive (XOR) and compatible (AND) binding was done using the SAPIN web framework [19]. SAPIN identifies the protein parts that could be involved in the interaction and provides template structures and then performs structural superimpositions to identify compatible and mutually exclusive interactions. We analyzed all complexes in the CORUM database with a complex size of less than 20 members. Each CORUM complex was broken down into combinations of three different proteins, and the respective protein sequences were used as the input for the SAPIN webserver. The workflow in SAPIN is:

1. Assigning all domains using Pfam (pfam_scan.pl with default parameter) based on the protein sequences.
2. Searching the 3DID [82] database to find potential domain-domain interaction hits of binary interaction partners provided.
3. Finding the best template of PPI structures (containing interacting domain A and domain B) by using the InterPreTS scoring function [83]. The interaction structures are evaluated by InterPreTS based on interface sequences aligned with 3Dstructures (we used MUSCLE [84] with default parameter). SAPIN selects one template that has more than 2.33 Z-score or the best score one among all candidates.
4. If two PPI structures share a same domain, structural superimpositions are performed based on the domain structure using Combinatorial Extension (CE [85]).
5. Analyzing for backbone clashes using FoldX [86] with superposed structures, and based on a backbone clashes threshold (15% of interface residues) the interactions are assigned compatible (AND) or mutually exclusive (XOR).
6. The reliability of the predictions with respect to sequence similarity to the template complex, we measured the sequence identity between the reference and homolog domains from the alignment based on the Needleman-Wunsch Algorithm [87]. Based on this, we determined a Z-score for the percentage of van der Waals backbone clashing among interface residues calculated for either AND or XOR, dependent on sequence similarity [19].

If a protein has at least two interacting partners, the domains mediating the interaction are superimposed on the reference domain, and the interacting domains are analyzed for compatibility (AND) or mutual exclusiveness (XOR). Note, that even two protein having similar domains do not necessarily bind at the same site/interface, but there are frequent cases of similar domains binding in a different way. As the best template suggestion based on sequence homology using INTERPRETS [9]. As the sequence is scored in INTERPRETS against a set of 1000 random sequences, the selection of the best template is not deterministic and can result in different results in different runs. We provide the average of the XOR/AND likelihood of independent runs in S4 Table.

Cell culture and differentiation of primary keratinocytes

For calcium-induced differentiation experiments, keratinocytes were seeded into 35 mm plates and grown in Keratinocyte Serum-Free Medium with supplements (KSFM; GIBCO) [3]. After reaching 70% confluence Keratinocyte Serum-Free Medium was exchanged for EMEM (Lonza) supplemented with 8% chelated FBS, EGF (10 ng/ml), 1% penicillin/streptomycin and 0.05 mM CaCl2. After 12 hour time point 0 h was collected, and the residual keratinocytes were synchronized for 2 h with EMEM containing 20% chelated FBS, EGF (10 ng/ml), 1% penicillin/streptomycin, and 0.05 mM CaCl2. After synchronization cells were washed once with PBS and cultured in EMEM, supplemented with 8% chelated FBS, EGF (10 ng/ml), 1% penicillin/streptomycin, and either 0.05 mM or 1.2 mM CaCl2, corresponding to non-calcium and calcium treatment, respectively. Cells were collected every 5 hours during a period of 45 hr.

Immunostaining and imaging

Keratinocytes were seeded onto 16-chambered LabTek slides (Nuncbrand). Kerationocyte differentiation was induced through addition of Calcium (as before), and sample were taken at the respective time points. Cells were fixed in 4% paraformaldehyde for 20 min and blocked with 4% BSA in PBS 1× (blocking solution). Primary staining was done using an antibody against Involucrin (Abcam, ab28057; dilution 1:1000 in blocking solution) followed by an Alexa Fluor 568 secondary antibody (Invitrogen Probes; dilution 1:200 in blocking solution). Cells were counterstained with DAPI (Sigma-Aldrich; concentration 1 μg/mL) and mounted using Mowiol solution. Staining was visualized on a Leica TCS SP5 confocal microscope with a 40× 1.25 NA objective at a zoom factor of 3 (1024 × 1024 pixels; 0.126 μm/pixel).