Figure 3. Deletion of Dnmt3a results in increased tumor heterogeneity, and upregulation of genes related to lipid metabolism.

(A) Schematic representation of FACS sorting strategy to isolate both RNA and DNA from Itga6^pos cells within the tumors. (B) Heatmaps representing gene expression (rlog transformed values) of the 391 differentially expressed genes between wild type and Dnmt3a-cKO sorted tumor cells. (C) Two-dimensional principal-component analysis (PCA) of RNA-seq samples from wild-type (n = 4) and Dnmt3a-cKO (n = 8) Itga6^bright sorted tumor cells. (D) Gene ontology analysis using Genomatix Online Software of the 114 downregulated and 277 upregulated genes in Dnmt3a-cKO tumors, divided by biological processes and over-represented signal transduction pathways. (E) Immunofluorescence staining for Krt14 and PPAR-γ of skin tumors from wildtype and Dnmt3a-cKO animals.

DOI: http://dx.doi.org/10.7554/eLife.21697.013

Figure 3—figure supplement 1. RNA samples submitted for sequencing were obtained from tumors scored predominantly as squamous cell carcinomas in wild-type and Dnmt3a-cKO mice.

(A) Hematoxylin/eosin staining from the four wild-type tumors used for RNA-seq. (B) Hematoxylin/eosin staining from the eight Dnmt3a-cKO tumors analyzed for RNA-seq. In A and B, immunofluorescence staining shown correspond to DAPI, ADFP (to ensure that no sebaceous adenomas were collected), and Krt14. (C) Representative hematoxylin/eosin staining and immunofluorescence of DAPI, ADFP and Krt14, of sebaceous adenomas eliminated from the RNA-seq study. Scale bar is 100 μm.

DOI: http://dx.doi.org/10.7554/eLife.21697.014

Figure 3—figure supplement 2. Loss of Dnmt3a results in a reduction of apoptosis in skin tumors.

(A) Representative images for TUNEL staining to detect apoptotic cells in skin tumors isolated from wild type and Dnmt3a-cKO animals. The right graph shows the quantification of the Tunel staining in wild type (n = 12) and Dnmt3a-cKO (n = 17) tumors. (B) Representative images for active Caspase-3 staining to visualize apoptotic cells in skin tumors isolated from wild-type and Dnmt3a-cKO animals. The right panel shows the quantification of the staining in wild-type (n = 6) and Dnmt3a-cKO (n = 6) tumors. Scale bar is 100 μm.

DOI: http://dx.doi.org/10.7554/eLife.21697.015

Figure 3—figure supplement 3. DMBA/TPA treatment induces an increase in cellular cell proliferation in Dnmt3a-cKO animals.

(A) Representative images of KI67 staining in treated or untreated back skin, and in skin tumors, of Dnmt3a-cKO and wild-type littermates. (B) Quantification of KI67 staining using the TMarker software, showing the percentages of KI67-positive cells in the different conditions studied and normalized to the proliferation in the interfollicular epidermis of wild-type mice. Scale bar is 100 μm.

DOI: http://dx.doi.org/10.7554/eLife.21697.017

Figure 3—figure supplement 4. Dnmt3a-KO tumors express high levels of PPAR-γ.

(A) CPM (Count per Million Read) values of the mRNA encoding for PPAR-γ obtained from the RNA-sequencing of the 12 tumors studied. (B) Representative immunofluorescence staining for DAPI, PPAR-γ and Krt14 in all the 12 tumors used for the RNA-sequencing experiment. Scale bar is 100 μm.

DOI: http://dx.doi.org/10.7554/eLife.21697.018