During tissue homeostasis stem cells are infrequently dividing; thus, and one of the characteristics of stem cells is their quiescence in situ. To determine the proliferative status of MHCI^- and MHCI⁺populations, we analyzed the expression of nuclear proliferation antigen Ki67, which is a marker for actively cycling cells. The data presented reflect keratinocyte proliferation since in normal epidermis, non keratinocytes are found to be non-cycling 2728. Although the absolute values of Ki67 may vary depending on the total number of gated cells, the ratio of Ki67 expressing MHCI^- and MHCI⁺ cells is held constant. Flow cytometric analysis showed that MHCI⁺ cells expressed more than four time higher levels of Ki67 than MHCI^- cells (Figure 1). Only 0.9% of MHCI^- cells expressed Ki67, while 3.9% of MHCI⁺cells were in the cell cycle. Given the low percentages of MHCI^-cells in the basal layer, it is clear that the bulk of cell production in the epidermis is accomplished through divisions of transient amplifying cells, a finding which is in accordance with the established view of epidermal homeostasis.

High proliferative potential is another feature of stem cells. One way of assessing proliferative potential of keratinocytes is to analyze their colony forming efficiency (CFE) 10. To assess proliferative potential of the two cell populations' colony forming efficiency (CFE) was analyzed in the primary and secondary cultures. Sorted cells were seeded at clonal densities and colonies formed evaluated after two weeks. In primary cultures, α6⁺/MHCI^- cells exhibited lower colony forming efficiency than α6⁺/MHCI⁺ cells (Figure 2). However, in secondary cultures α6⁺/MHCI^- showed higher CFE than α6⁺/MHCI⁺ cells. From primary to secondary culture, an increase of 38 times was observed in the CFE of α6⁺/MHCI^- cells, while the CFE of α6⁺/MHCI⁺ cells increased only 3.6 times (Figure 2). Previously, lower initial CFE was observed in limbal epithelial stem cell 29. Our data indicate that α6⁺/MHCI^- cells have higher proliferative potential than α6⁺/MHCI⁺ cells, another characteristic attributed to stem cells. The majority of available data regarding CFE are from secondary or tertiary keratinocyte cultures 1030. In the secondary cultures, α6⁺/MHCI^- cells did display higher CFE than α6⁺/MHCI⁺ cells, which is an indication of a higher proliferative potential of the α6⁺/MHCI^- cells 10. The lower CFE of α6⁺/MHCI^- cells compared to α6⁺/MHCI⁺ cells that we recorded in our primary cultures may be due to the time needed for stem cells to transit from a quiescent to a proliferative state.

Microarray profiles of stem cells and their progeny provide a global view into differences of expression of a large number of genes and enable analyses of molecular processes involved in self renewal, proliferation and differentiation. Gene expression profiles of hair follicle bulge stem cells were recently reported 921313233, yet until now no data are available with regard to human interfollicular keratinocyte stem cells. We report on transcriptional profiles of putative human keratinocyte stem cells and their immediate progeny, transient amplifying cells. Global gene expression profile was obtained from sorted α6⁺/MHCI^- cells and α6⁺/MHCI⁺ cells using DNA microarray chips. We identified a comprehensive list of differentially expressed genes. Notably, all of the MHCI genes were downregulated in α6⁺/MHCI^- cells, thus confirming the successful separation of α6⁺/MHCI⁺ and α6⁺/MHCI^- cells. The data also show that expression of MHCI proteins in keratinocytes is regulated at the transcriptional level. The HLA-E transcript is downregulated in α6⁺/MHCI^- cells confirming our previous results obtained at the protein level 22. The expression of non-classical HLA molecules is thought to protect cells that lack classical HLA expression from lysis by NK cells. At present, it is not known what mechanisms protect presumptive stem cells, MHCI^- cells, from attack by NK cells, especially since MHCI^- cells do not express detectable levels of non-classical HLA-E and HLA-G molecules 22.

We found that most of the mRNAs of genes encoding cellular receptors and other cell surface molecules were more abundant in α6⁺/MHCI^- cells than in α6⁺/MHCI⁺ cells (see Additional file 1). Conversely, mRNAs of genes encoding proteins that take part in ribosome biosynthesis, RNA splicing, translation, protein degradation, and energy metabolism were more abundant in α6⁺/MHCI⁺ cells (see Additional file 1). These findings are consistent with reports by other investigators who demonstrated that stem cells are characterized by few ribosomes and mitochondria (features related to undifferentiated state of stem cells) but contain a large numbers of receptors 934.

In reference to stem cells quiescence, it should also be noted that transcripts of CDKN1C and CDKN2A whose products are cyclin-dependent kinase inhibitors were enriched in α6⁺/MHCI^- cells (Figure 3A). The product of CDKN1C, p57^Kip2, one of the Cip/Kip family members, is tightly associated with inhibition of proliferation of human interfollicular keratinocytes 35, and is upregulated in hair follicle bulge 31, while the product of CDKN2A, a 16 kDa protein p16^INK4a imposes a G1 cell cycle arrest 3637. Furthermore, in addition to the low protein level of Ki67 observed in MHCI negative population (Figure 1), the transcript of Ki67 was less abundant in α6⁺/MHCI^- cells (Table 1, and Additional file 1). Conversely, transcripts of genes encoding proteins that are related to cell proliferation such as cyclins, proteins involved in chromosome remodeling, DNA replication and repair were preferentially enriched in α6⁺/MHCI⁺ cells (Table 1, and Additional file 1).

Interestingly, type I IFN (IFN-α, and IFN-β) has been shown to inhibit cell proliferation by inducing G1 cell cycle arrest. It has been reported that interferon α (IFN-α) has antiproliferative effects on bone marrow stromal precursors, hepatic progenitor cells, and mesenchymal stem cells 394041. We observed enrichment of transcripts of the IFN-α family of proteins in α6⁺/MHCI^- cells as well as STAT2, the specific transducing activator of IFN-α transcription. This is the first report that suggests involvement of type I IFN in epidermal stem cell quiescence. Further studies are needed to determine whether IFN-α is synthesized by α6⁺/MHCI^- cells, whether its pathway is active and whether it contributes to α6⁺/MHCI^- cell quiescence (Table 1).

It has been reported that the components of the inositol phospholipid signaling system are present and that the system itself is active in murine embryonic stem cells 42. In the present study, we demonstrate that several components of the inositol phospholipid signaling system are enriched in α6⁺/MHCI^- cells (Table 1). Nevertheless, the functional significance of this observation needs further investigation.

Among the transcripts enriched in α6⁺/MHCI^- cells, there were transcripts previously shown to be upregulated in cell population enriched for human interfollicular stem cells, such as α6 integrin 11, melanoma-associated chondroitin sulfate proteoglycan 43, phosphorylase kinase α2 43, and the transcript of p53 homolog p51/p73L/p63/p40 44 (Figure 3A and Additional file 1). Using flow cytometry we found that MHCI^- cells expressed lower level of transferrin receptor (CD71), a negative marker used to enrich for interfollicular epidermal stem cells 11, when compared to MHCI⁺ cells (Figure 4). It should also be noted that although differences in the expression of CD71 transcript were lower than 2 fold, CD71 mRNA was downregulated in α6⁺/MHCI^- cells compared to α6⁺/MHCI⁺ cells consistently in both arrays (see Additional file 2).

Among the transcripts enriched in α6⁺/MHCI⁺ cells, there were mRNAs of genes whose products are expressed at low levels in cell population enriched for human interfollicular stem cells, such as desmosomal proteins including desmoglein 3 (DSG3) 14, as well as the proliferation associated transcription factor c-Myc, found to be expressed at the lower levels in cultured human interfollicular stem cells 45 (Figures 3A, 3B and Additional file 1). Moreover, it has been reported that epidermal growth factor receptor (EGFR) signaling is downregulated in putative human interfollicular stem cells 43. In accordance with this observation, we found that EGFR itself was downregulated in α6⁺/MHCI^- cells (Fig. 3A and Additional file 1).

Most notably, we present results that demonstrate that putative stem cells have lower expression of mRNAs encoding proteins that take part in energy metabolism, which can explain how stem cells can be quiescent and at the same time maintain small size (see Additional file 1).

We compared α6⁺/MHCI^- transcriptional profile with the published transcriptional profile of human hair follicle stem cells 32 and with four different sets of data of murine hair follicle stem cells 9213133. We found that eleven genes, which were enriched in human hair follicle stem cells, were also enriched in α6⁺/MHCI^- cells (Table 1). Similarly, mRNAs of nine genes, which were downregulated in human hair follicle stem cells, were also downregulated in α6⁺/MHCI^- cells (Table 2). Nevertheless, the differences between the gene expression profiles of stem cells from the two human tissues, i.e. interfollicular and follicular, were also observed including the genes that demonstrated two fold difference in the expression (total of nine genes) (see Additional file 3).

mRNAs of eighty-two genes, which were enriched in murine hair follicle stem cells, were also enriched in α6⁺/MHCI^- cells, while mRNAs of forty-one genes, which were downregulated in murine hair follicle stem cells, were also downregulated in α6⁺/MHCI^- cells (Tables 1, 2 and Additional file 4). Transcription factors LHX2 and TCF3 that were shown to maintain SC features 3346, were among the genes that were upregulated in α6⁺/MHCI^- cells as well as in murine hair follicle stem cells. Upon screening of our microarray database for TCF3 targets 46, we found that transcripts of twelve genes reported to be upregulated by TCF3 were more abundant in α6⁺/MHCI^- cells and conversely transcripts of nine genes repressed by TCF3 were more abundant in α6⁺/MHCI⁺ cells (see Additional file 5). Interestingly, both arrays that we performed showed that LHX2 was among the most upregulated mRNAs in α6⁺/MHCI^- cells, while WIF-1 mRNA, which was the most enriched mRNA in murine hair follicle stem cells according to one report 33, was the mRNA that showed the highest difference of expression between α6⁺/MHCI^- cells and α6⁺/MHCI⁺ cells (97 fold).

It has been shown that TGF-β and the bone morphogenic factors are upregulated in epidermal stem cells 93133. Consequently, hair follicle stem cells are enriched with TGF-β/BMP targets 9. In accordance with those findings, we observed that transcripts of several genes whose products are necessary for the activation of TGF-β/phospho-Smad pathway, such as genes necessary for latent TGF-β activation (LTBP-1 and LTBP-2), secreted activators (BMP5, BMP8, BMP10, BMP15), and transcriptional activators of TGF-β responses (MADH3 and MADH6), were enriched in α6⁺/MHCI^- cells compared to α6⁺/MHCI⁺ cells (Table 1). In addition, transcripts of forty-nine target genes shown to be upregulated by TGF-β/BMP pathway were more abundant in α6⁺/MHCI^- cells. Conversely, transcripts of the genes whose expression is shown to be suppressed by TGF-β/phospho-Smad pathway, including transcription factors c-Myc and KLF4, were more abundant in α6⁺/MHCI⁺ cells (Figure 3B, Table 2). Since TGFβ/BMP pathway is tightly associated with stem cell quiescence 314748, upregulation of BMPs in α6⁺/MHCI^- cells might explain why these cells exhibit characteristics of quiescent cells.

Wnt pathway plays an important role in hair follicle morphogenesis and cycling 495051. Researchers found that transcripts of several Wnt genes were downregulated in the mouse hair follicle bulge stem cells. In addition, higher levels of several genes that inhibit Wnt signaling pathway as well as higher levels of transcripts of the Wnt receptors were found in epidermal stem cells 9213233. In the same cells, in general, targets of Wnt signaling (such as hair keratin, KRTHA1, nuclear proliferation antigen Ki67 93351 are downregulated. Consistent with these observations, we found that WNT3 and WNT4 were downregulated, while Wnt receptors FZD1, FZD4, FZD7, and the inhibitors of Wnt signaling pathway, DAB2, TCF3, CTBP2, WIF1, DKK1, and DKK2 were upregulated in α6⁺/MHCI^- cells (Table 1 and 2). Furthermore, transcripts of thirty eight genes that are known to be upregulated by Wnt signaling pathway were less abundant in α6⁺/MHCI^- cells, including MYCBP and type I hair keratin 1 (KRTHA1) (Figure 3B and Table 2). Also as expected to be found in stem cells, transcripts of genes that are downregulated by Wnt signaling pathway were found to be more abundant in α6⁺/MHCI^- cells. We found transcripts of fourteen such genes in α6⁺/MHCI^- cells (Table 2).

Stem cells are the least differentiated cells in their tissue of origin; therefore, transcription factors and signaling pathways that induce differentiation are expected to be downregulated in these cells. As mentioned above, we found that MYC was less abundant in α6⁺/MHCI^- cells compared to α6⁺/MHCI⁺ cells (see Additional files 1 and 6). Upon screening of our microarray database for c-Myc targets, we found that the transcripts of sixty-one genes including MYCBP (Figure 3B and Additional file 6), which were reported previously to be upregulated by c-Myc, were upregulated in α6⁺/MHCI⁺ cells. Conversely, transcripts of nineteen genes that were reported to be downregulated by c-Myc were upregulated in α6⁺/MHCI^- cells (see Additional file 6). Since many targets of c-Myc are involved in the ribosomal biogenesis, the downregulation of MYC may account for the observed downregulation of large numbers of genes that are involved in ribosomal biogenesis in α6⁺/MHCI^- cells (see Additional file 1), and may be an additional evidence that the downregulation of MYC in stem cells is related to their quiescent/undifferentiated state.

Similar to c-MYC expression, the expression of KLF4 (Kruppel-like factor 4), a transcription factor that is mainly expressed in the differentiating layers of epidermis 52, and BMP2, a member of bone morphogenetic protein family that is mainly expressed in proliferative basal and differentiated suprabasal keratinocytes 53, were downregulated in α6⁺/MHCI^- cells compared to α6⁺/MHCI⁺ cells (Table 2 and Additional file 1).

Notch1 signaling has been shown to stimulate differentiation in mammalian skin 54. Transcripts of the genes, such as fillagrin, integrin alpha 6, loricrin, (FLG, INTA6, LOR) whose expression is repressed by Nothch1 signaling 5455 were more abundant in α6⁺/MHCI^- cells (Figure 3A, and Additional file 1). Furthermore, expression of KRT5, which is upregulated by Notch1 signaling 54, was more abundant in α6⁺/MHCI⁺ cells (Figure 3A, and see Additional file 1). We investigated whether the levels of Notch1 activity differ in α6⁺/MHCI^- and α6⁺/MHCI⁺ cells. By using flow cytometry analysis with an antibody specific for cleaved/active Notch1, we could not detect any significant presence of cleaved/active Notch1 in α6⁺/MHCI^- cells contrary to α6⁺/MHCI⁺ cells (Figure 5), which indicates that differentiation-inducing pathway Notch1 is downregulated/inhibited in α6⁺/MHCI^- cells.

It has been shown that Notch1 signaling pathway activates NF-κB pathway and upregulates subunits of NF-κB and its targets 565758. Thus, we investigated whether the levels of activity of NF-κB pathway, which is downstream of Notch1 pathway, differ in MHCI^- and MHCI⁺ cells. First, we analyzed the expression of NF-κB subunit RelA in MHCI^- and MHCI⁺ cells and found a positive correlation between the expression of NF-κB subunit RelA and MHCI (Figure 6). Since both MHCI molecules and NF-κB subunits are targets of NF-κB pathway, this result suggested that similar to the Notch1 pathway, NF-κB pathway is downregulated in MHCI^- cells.

Search of our cDNA array data base for activators and targets of NF-κB pathway identified transcripts of genes, which either directly activate NF-κB pathway, or play a role in the activation of this pathway, such as BMP2 and MALT1 59, that were more abundant in α6⁺/MHCI⁺ cells compared to α6⁺/MHCI^- cells. Furthermore, in accordance with this finding, transcripts of thirty genes that are reported to be upregulated by Rel/NF-κB transcription factors 59, were enriched in α6⁺/MHCI⁺ cells (Table 2). Thus, our microarray data suggest that the NF-κB pathway is downregulated in α6⁺/MHCI^- cells as compared to α6⁺/MHCI⁺ cells. To determine whether NF-κB pathway is indeed more active in α6⁺/MHCI⁺ cells, we performed TRANS AM NF-κB ELISA assay using nuclear extracts of α6⁺/MHCI⁺ and α6⁺/MHCI^- cells. We found that the relative amount of nuclear phospho-NF-κB p50 bound to DNA, an indicator of an active NF-κB pathway, is higher in α6⁺/MHCI⁺ cells than α6⁺/MHCI^- cells (Figure 7). Collectively, the data demonstrate that Notch1 signaling as well as the downstream NF-κB pathway are both downregulated in α6⁺/MHCI^- cells compared to α6⁺/MHCI⁺ cells.

To gain further insights of epidermal differentiation, we compared our database with the published database of differentially expressed genes in basal and suprabasal layers of the human epidermis 60. The comparison of our data with the transcriptional profile of the genes that are differentially expressed in the basal and differentiated layers of the epidermis suggests that TGF-β/phospho-Smad pathway-induced transcription profile fades along the epidermal differentiation axis. The abundance of transcripts, which are upregulated by TGF-β/phospho-Smad pathway (such as COL6A1, LTBP2, MMP9, PLAT), decrease during differentiation, from presumptive stem to transient amplifying cells (basal layer) and further to cells of the suprabasal layers, where these transcripts are present at the lowest level. In addition, the transcript of MADH3, a transcriptional activator of TGF-β responses, is also increasingly downregulated. Conversely, abundance of transcripts, which are downregulated by TGF-β/phospho-Smad pathway (such as KLF4 and MYC), appear to positively correlate with the increase of epidermal differentiation (Figure 8A).

On the other hand, the comparison suggests that Notch1 and Wnt pathway-induced transcription profiles strengthen along the epidermal differentiation axis. Transcripts of MYC and ELF1, which are upregulated by Wnt signaling gradually increase. These findings are in accordance with the previous reports, which demonstrate that TGF-β/phospho-Smad pathway prevents keratinocyte differentiation 48, and, conversely, Notch1 and Wnt pathways induce keratinocyte differentiation 546162. Thus, it might be possible that while TGF-β/phospho-Smad pathway is gradually downregulated, Wnt pathway becomes increasingly more active during epidermal differentiation. Nevertheless, further investigation is necessary to validate this hypothesis. Similarly, while the transcription of KRT1, which is upregulated by Notch1 signaling, increases, ITGA6 mRNA, which is downregulated by Notch1 signaling, decreases in keratinocytes during differentiation (Figure 8A). Several reports demonstrated that suprabasal cells have higher Notch1 activity than basal cells 5563. As already mentioned, employing flow cytometry analysis and antibodies against cleaved/active Notch 1, no detectable levels of cleaved/active Notch 1 was observed in MHCI^- cells indicating the lack of Notch1 activity (Figure 5).

Interestingly, previous reports suggested that strong adherence of stem cells to extracellular matrix-rich basement membrane may be involved in retaining these cells in their natural residence (niche) 5264. In accordance with these observations, the comparison of our data with published transcription profiles of the basal and suprabasal cells of the epidermis revealed that during differentiation transcripts of several genes (MUC1, COL6A1, ITGA6, MMP9, PLAT, CEACAM1) whose products are soluble or membrane-bound factors that play a role in the interaction of cells with the microenvironment, decrease gradually during differentiation (Figure 8A).

We also found that twenty-one genes were upregulated in α6⁺/MHCI⁺ cells (TA cells) alone, and later become downregulated during terminal differentiation (Figure 8B). Among these transcripts there are ones related to cell cycle (CCNB1, CCND2, RFC5) as well as mRNAs of the genes whose products induce cell growth and division (ZWINT, BLCAP). Since α6⁺/MHCI^- cells are quiescent, and terminally differentiating cells are post-mitotic it is not surprising to find transcripts whose products accelerates cell proliferation among the genes that are upregulated only in α6⁺/MHCI⁺ cells during epidermal differentiation. Similarly, genes whose products suppress cell growth and proliferation were enriched both in α6⁺/MHCI^- cells and terminally differentiating suprabasal cells, such as PLAGL1, a zinc finger transcription factor that induces cell cycle arrest in the skin and whose expression is diminished in basal cell carcinomas 65, putative tumor suppressors insulin-like growth factor-binding protein 7 (IGFBP7), and DOC1 6667 (see Additional file 1 and reference 60).

Neonatal foreskins were obtained from routine circumcisions. After washing in PBS, and removing of subcutaneous fat, the tissue was cut into 5 × 5 mm pieces and incubated overnight at 4°C in Dulbecco's modified Eagle's medium containing 2.5 mg/ml Dispase II (Boehringer Mannheim, Indianopolis, IN), penicillin (100 units per ml), and streptomycin (100 μg/ml). Epithelial sheaths were separated from the dermis by gentle peeling with forceps. Keratinocytes were harvested after incubation with trypsin/EDTA solution (0.05% and 0.01%, respectively) for 10 minutes at 37°C 22.

After trypsin neutralization and blocking with buffer containing BSA and human IgG, keratinocytes were immunolabeled with R-phycoerythrin (PE)-conjugated mouse anti-human β2 microglobulin (BD Biosciences, San Diego, CA, USA), and with a monoclonal anti-human α6 integrin-fluorescein isothiocyanate (FITC) conjugate (Serotec, Oxford, UK). Control samples were incubated with appropriate isotype controls. All incubations were performed at 4°C. Cells were sorted using FACSVantage, or FACSAria (Becton Dickinson, Franklin Lakes, NJ). Flow cytometry data used for sorting cells that donated RNA for microarray experiments, as well as representative controls are shown in Additional file 7. The data were analyzed using Cell Quest software (BD Biosciences). Selection of basal keratinocytes with anti-human α6 integrin eliminates suprabasal cells (terminally differentiated keratinocytes), and non-keratinocytes, such as melanocytes and dentritic cells) 2160.

For the expression of Ki67 isolated keratinocytes were immunolabeled with mouse anti-human antibody against MHCI (BD Biosciences) and with a polyclonal antibody against Ki67 (Zymed San Francisco, CA). Secondary antibodies were goat anti mouse IgG1 PE-conjugated antibody (Southern Biotechnology Associates Inc. Birmingham, AL) and donkey anti rabbit FITC-conjugated antibody. For the setting of gates, secondary control and single color positive controls were used.

Keratinocytes were grown in a keratinocyte medium (3:1 DMEM, F12) supplemented with FBS and additives in 100 mm culture dishes, previously seeded with lethally irradiated 3T3 fibroblasts 68. The medium was replaced every other day. Colonies were visualized after two weeks in culture following fixation in 10% formalin and staining with 2% rhodamine B (Sigma). Colony forming efficiency (CFE) is the ratio of colony number to plating cell number expressed as a percentage. Results are presented as means ± SD.

Following cell sorting, RNA isolation (RNeasy Mini Kit (Qiagen)) and amplification (MessageAmp aRNA Kit (Ambion)) microarray was performed using Affymetrix HGU 133 A+B GeneChip set. Scanned images of Affymetrix GeneChip arrays were quantified using Affymetrix GCOS software, Gene Chip Operating System. The target intensity was set to 500 and the default parameters were used. The results were filtered and probe sets with a "No Change" (NC) call were removed. Additionally, probe sets that were scored "Increased" (I) or "Marginal Increase" (MI), but called absent on the experimental sample, as well as probe sets that were scored Decreased (D) or "Marginal Decrease" (MD) and called absent in the baseline sample, were removed. For the resulting list of probe sets a fold change column was calculated. Microarray Gene Expression Data have been deposited, accession number GSE11089 69. For the genes that are differentially expressed in both arrays see Additional files 2, 8 and 9.

Total RNA was isolated directly after cell sorting using the RNeasy Mini Kit (QIAGEN) according to manufacturer's protocol. Extracted RNA was reverse transcribed by using Sensiscript RT Kit (QIAGEN) according to manufacturer's instructions. It must be noted that RNA used for RT-PCR and for each microarray analysis was isolated from cells derived from multiple skin samples that consisted of different donors. By pooling skin samples we were able to obtain sufficient amount of cells and at the same time average any potential individual differences. Primers for KRTHA1 70, β-catenin 71, KLF4 72, and DSG3 73 were published previously. Other primers used are: ITGA6 F: 5'-TGCTGTTGGTTCCCTCTCAGAT-3'. ITGA6 R: 5'-CTGGCGGAGGTCAATTCTGT-3'. MYCBP F: 5'-ATGGCCCATTACAAAGCCGC-3'. MYCBP R: 5'-CTATTCAGCACGCTTCTCCT-3'. Initial PCR step was 1 minute at 94.0°C, followed by 25, 30, 35 cycles of a 15 seconds melting at 94.0°C, a 15 seconds annealing at 55.0°C and a 15 seconds extension at 72.0°C. The final extension was at 72.0°C for 1 minute.

For the analyses of protein expressions in MHCI^- and MHCI⁺ cells the following antibodies were used: Antibody against MHCI, mouse anti-human (IgG1 isotype, BD biosciences); antibody against NF-κB p65, rabbit anti human (Santa Cruz); antibody against cleaved/active Notch1, rabbit anti human (Calbiochem); and antibody against CD71, mouse anti human (IgG1 isotype, Diaclone, France). Secondary antibodies used for these analyses were: FITC-conjugated goat anti-mouse IgM (Sigma), PE-conjugated goat anti mouse IgG1 (SouthernBiotechnology, Birmingham, Al), and FITC-conjugated donkey anti-rabbit (Jackson ImmunoResearch Lab. Inc., West Grove, PA).

Nuclear extracts were obtained using Nuclear Extract Kit purchased from Active Motif (Carlsbad, CA). 100,000 cells (α6⁺/MHCI⁺ and α6⁺/MHCI^- each) were directly sorted in PBS buffer that contained phosphatase inhibitors, supplied with the kit. Levels of the active/phospho-NF-κB p50 in the nucleus were assayed using TransAM NF-κB p50 Transcription Factor Assay Kit (Active Motif) according to manufacturer's protocol. Nuclear extract of HeLa cells stimulated with TNF-α for 30 minutes supplied by Active Motif was used as a positive control.

Additional file 1 contains all the transcripts, which demonstrated equal or higher than 2 (≥ 2) fold difference between α6⁺/MHCI^- cells and α6⁺/MHCI⁺ cells (Additional file 1 shows only values that are ≥ 2 fold in log2 scale). The genes that are consistently downregulated or upregulated in both arrays were shown in tables S6, S7 and S8. All transcripts that belong to MHCI protein family are downregulated in α6⁺/MHCI^- cells as expected, which indicate that our selection process was successful (see Additional file 1). Several reports were used as the base to screen for c-Myc target genes 7475767778798081, Wnt target genes 828384858687888990919293, TGF-β/BMP target genes 94959697, and targets of NF-κB pathway 599899100.

Student's t-test was applied for statistical analysis. Error bars represent ± SD.