Figure 1. Growth characteristics of GLI2 expressing cells in monolayer culture (A) Expression of GLI2 and downstream targets GLI1 and PTCH in control

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1 Figure 1. Growth characteristics of GLI2 expressing cells in monolayer culture (A) Expression of GLI2 and downstream targets GLI1 and PTCH in control HaCaT Tet, uninduced HaCaT GLI2 and induced HaCaT GLI2 cells. For each cell type, cells were harvested from a T75 flask and RNA was isolated using TRIZOL reagent. Expression levels were measured relative to GUSB by quantitative RT PCR using the ABI Assays-on-Demand as described previously (Snijders et al., 2005). (B) Growth characteristics in monolayer cultures of inducible HaCaT GLI2 cells with and without addition of doxycycline to the medium. Five hundred HaCaT GLI2 cells were seeded in 100 μl culture media in 12 wells of tissue culture treated 96-well plates. After allowing cells to attach and grow for 24 hours, an additional 100 μl of culture media was added and the media in six wells of each plate was supplemented with 1 μg/ml doxycycline to induce GLI2 expression. Thereafter, one plate was harvested each day over a four day period, stored at -80 C, and then proliferation was measured using the CyQUANT Cell Proliferation Assay kit. (C) Growth characteristics in monolayer cultures of HaCaT cells infected with p-babe-6xhis- GLI2 N.

2 Figure 2. GLI2 induces genome instability (A) Methotrexate resistant colonies arising in HT1080 cells and derivatives expressing egfp, CCND1 or 6xHis-GLI2ΔN. To prepare HT1080 cells overexpressing CCND1 or GLI2ΔN, the genes were cloned into FG12-RFP or FG12-eGFP lentiviral vectors, respectively. The parental HT1080 cells were subsequently infected with these lentiviruses or the FG12-eGFP empty vector lentivirus to obtain control lentivirus infected HT1080 cells (HT1080 egfp). Approximately 5 days post infection, 1x10 3 cells were seeded in each well of two 6-well plates and the cells expanded to ~80% confluency before 4x10 5 cells from each well were cultured in medium containing 25 nm methotrexate (2-3x LD50). Plates were incubated for 27 days after which time one resistant colony was picked from each 100 mm plate and expanded in medium containing 25 nm methotrexate. The colonies remaining on the plates were fixed in methanol/acetic acid (3:1) and stained with crystal violet. Colonies visible by eye (> ~50 cells) were counted as described previously (Snijders et al., 2003; Snijders et al., 2008). Shown are boxplots representing the number of resistant colonies. The thick horizontal line represents the median number of colonies, while the bottom and top of each box represent the 25 th and 75 th percentile, respectively. The width of each box is proportional to the square root of the number of samples. Outlier values are indicated with circles. (B) The p-values for each pairwise comparison are shown and were calculated using a two-sided Wilcoxon rank sum test. A p-value cut-off of 0.05 was used to declare significance. The GLI2 and CCND1 expressing HT1080 cells gave rise to equal numbers of drug resistant colonies and these numbers were significantly greater than the number recovered from either egfp expressing or parental HT1080 cells. Note, a significantly greater number of colonies was also formed in the egfp expressing HT1080 cells compared to parental HT1080 cells, from which we did recover a few drug resistant colonies unlike previous studies (Paulson et al., 1998). (C) Heatmap representation of copy number changes detected by array CGH in methotrexate resistant colonies. For array CGH, cells were harvested from a T75 flask and DNA extracted by overnight incubation at 55 C in a 3 ml solution containing 0.01 M Tris, ph 7.5, M EDTA, ph 8, 0.5% SDS and 0.1 μg/μl proteinase K. The DNA was precipitated with ethanol, recovered it by spooling and then dissolved it in ~300 μl H 2 O. Labeling of DNA for array CGH, hybridization and image analysis were carried out as described previously (Snijders et al., 2003). Each column represents one resistant colony. Individual BAC clones are shown as rows and ordered according their genome position (May 2004 freeze, UCSC Genome Browser). Copy number aberrations were assigned as described previously (Fridlyand et al., 2006). Losses are indicated in red, gains in green and amplifications as yellow dots. Copy number changes present in the parental HT1080 cell line include +3q, +5p and deletion of a single BAC at 9p21 spanning CDKN2A. Array CGH data were deposited in the NCBI Gene Expression Omnibus database, accession number GSE No significant differences were observed between the various HT1080 genotypes in the spectra of copy number changes or the number and types of copy number alterations arising in the drug resistant cells

3 Figure 3. Contraction of HaCaT Tet and HaCaT GLI2 organotypic cultures In organotypic cultures, fibroblasts are initially cultured for a week in collagen gels resulting in contraction of the collagen gel to form a cup. Keratinocytes are then added on top of the collagen/fibroblast layer and the co-cultures further propagated for four days, before exposure to air. A stratified and differentiated epithelium is formed within three weeks. (A) Photomicrographs of reconstructs prior to fixation. Reconstructs were placed on a light box and photographed from the top. Contraction was determined by measuring the surface area of the mesa-shaped plateau formed by the fibroblast/collagen matrix (dashed black line). The surface area occupied by HaCaT cells identified as the embossed and opaque appearing area was also measured, which in control HaCaT Tet and HaCaT GLI2 cells (-doxycycline) was identical to the fibroblast area. In GLI2 expressing HaCaT GLI2 cells (+ doxycycline), however, the HaCaT area was smaller as indicated by the yellow dashed line. (B) Measured surface areas of fibroblast and HaCaT regions. Measurements were made from four replicate cultures for each cell type. These cultures used foreskin fibroblasts from at least two different donors. To control for variations in contraction capacities of different fibroblast cultures, fibroblast and HaCaT surface area calculations were each normalized to the fibroblast or HaCaT surface areas of reconstructs of HaCaT Tet cells, respectively.

4 Figure 4. Characteristics of organotypic cultures of primary foreskin derived keratinocytes grown on a collagen matrix containing foreskin derived primary fibroblasts Antibody staining patterns in sections from organotypic cultures of primary foreskin keratinocytes and fibroblasts visualized by immunohistochemistry (IHC) or immunfluorescence (IF). Sections were processed as for HaCaT cell cultures and the analyses were carried out with at least two pairs of fibroblasts and keratinocytes from two different donors.

5 Figure 5. Overexpression of GLI2 alters expression of genes related to the identification, growth and differentiation of stem cells. (A) Fold induction (values >1) and fold repression (values <1) after induction of GLI2 in organotypic cultures of GLI2 expressing HaCaT GLI2 and control HaCaT Tet keratinocytes. After epidermalization of the doxycycline induced HaCaT GLI2 (n=2) and control HaCaT Tet (n=2) organotypic cultures, the epidermal layer was carefully peeled away from the underlying fibroblast containing dermal equivalents using needle nose forceps. The epidermal layer was placed in 600 µl of TRIZOL reagent and stored at -80 C until RNA was isolated. Expression levels were measured relative to GAPDH by quantitative RT PCR using the Human Stem Cell RT 2 Profiler PCR Array (SuperArray Bioscience). Genes were considered not expressed in the event that one sample within either HaCaT GLI2 or HaCaT Tet duplicates failed to detect expression or when the Ct for either or both samples within each duplicate exceeded 30 cycles. Relative expression levels of duplicate cultures were averaged and fold induction/repression was calculated by dividing the average relative expression level in HaCaT GLI2 cells by the average relative expression level in control HaCaT Tet cells. (B) Expression of genes (n=7) relative to GAPDH that were induced in GLI2 expressing HaCaT GLI2 organotypic cultures, but not detected in organotypic cultures of control HaCaT Tet cells.

6 Figure 6. Induction of SOX2 expression in primary keratinocytes and HaCaT GLI2 cells. (A) Expression of GLI2, downstream target GLI1 and putative downstream target SOX2 in primary foreskin derived keratinocytes (FK5) infected with a GLI2 retrovirus or empty vector control. RNA was isolated using TRIZOL reagent. Expression levels were measured relative to GUSB by quantitative RT PCR using the ABI Assays-on-Demand as described previously (Snijders et al., 2005). (B) Response of GLI2 target genes GLI1 and PTCH1 and putative downstream target SOX2 after induction of GLI2 in HaCaT GLI2 cells. HaCaT GLI2 cells were seeded in six 6-well plates (200,000 cells per well; 4 wells per plate). Cells were allowed to recover overnight after which time RNA was harvested from one plate (T=0 hr). GLI2 expression was induced in the remaining plates by replacing the media in three wells of each plate with media containing doxycycline (1 µg/ml). Plates were harvested at 3, 6, 9 and 12 hours post addition of doxycycline. RNA was isolated using TRIZOL and expression levels were measured relative to GUSB by quantitative RT PCR using the ABI Assays-on-Demand. Relative expression levels for each gene were normalized to the average expression level of uninduced samples. The standard deviation represents the variation in gene expression between independent wells at each time point.

7 Figure 7. Smooth muscle actin staining in oral SCC with GLI2 amplification Antibody staining patterns of smooth muscle actin (SMA) in sections of two oral SCC showing highlevel amplification of the GLI2 locus. Strong staining was observed in areas surrounding epithelial tumor nests similar to the SMA staining pattern in GLI2 expressing HaCaT GLI2 reconstructs

8 Figure 8. Introduction of a layer of acellular collagen separating the dermal and epithelial compartments in organotypic cultures does not affect differentiation of uninduced HaCaT GLI2 cells, but prevents transdifferentiation of fibroblasts in GLI2 expressing HaCaT GLI2 cells. To create organotypic cultures with an acellular layer of collagen separating the epithelial and collagen/fibroblast layers, collagen solution (75 μl) was pipetted into the middle of each reconstruct after the fibroblasts had contracted the collagen layer to form the mesa shaped plateau. A cloning ring was immediately placed into the collagen and the plate was incubated at 37 C for 15 minutes. An additional 100 μl of collagen solution was pipetted into each cloning ring and the plates incubated at 37 C for 30 minutes before pipetting 250,000 egfp labeled HaCaT GLI2 cells onto the collagen layer and further culturing the reconstructs in the usual manner. (A) Formalin fixed paraffin embedded sections of cultures of uninduced HaCaT GLI2 cells were stained with antibodies to cytokeratin 10/13, involucrin and integrin β4 and visualized using Alexa 594 labeled secondary antibodies (red) as described in Table S5. Nuclei were counterstained with DAPI (blue). The acellular collagen layer is indicated by the arrowheads. (B) Sections from reconstructs prepared with a layer of acellular collagen separating the dermal and epithelial compartments of egfp expressing HaCaT GLI2 cells with or without addition of doxycycline (right and left panels, respectively). The acellular collagen layer appears as a clear region under the epithelial cells in the H&E stained sections (top panels). Sections from reconstructs of control and GLI2 expressing HaCaT GLI2 cells (left and right bottom panels, respectively) stained for egfp (green) and SMA (red).

9 Figure 9. Invading epithelial cells down regulate expression of the GLI2 target gene, BCL2. Sections of organotypic cultures of GLI2 expressing HaCaT GLI2 cells stained using HRP and DAB detection and antibodies to BCL2 (middle panel). The locations of keratinocytes in the sections are indicated by staining adjacent sections with antibodies to CAM5.2 and AE1/AE3.

10 REFERENCES Fridlyand, J., Snijders, A. M., Ylstra, B., Li, H., Olshen, A., Segraves, R., Dairkee, S., Tokuyasu, T., Ljung, B. M., Jain, A. N., et al. (2006). Breast tumor copy number aberration phenotypes and genomic instability. BMC Cancer 6, 96. Paulson, T. G., Almasan, A., Brody, L. L., and Wahl, G. M. (1998). Gene amplification in a p53- deficient cell line requires cell cycle progression under conditions that generate DNA breakage. Mol Cell Biol 18, Snijders, A. M., Fridlyand, J., Mans, D. A., Segraves, R., Jain, A. N., Pinkel, D., and Albertson, D. G. (2003). Shaping of tumor and drug-resistant genomes by instability and selection. Oncogene 22, Snijders, A. M., Hermsen, M. A., Baughman, J., Buffart, T. E., Huey, B., Gajduskova, P., Roydasgupta, R., Tokuyasu, T., Meijer, G. A., Fridlyand, J., and Albertson, D. G. (2008). Acquired genomic aberrations associated with methotrexate resistance vary with background genomic instability. Genes Chromosomes Cancer 47, Snijders, A. M., Schmidt, B. L., Fridlyand, J., Dekker, N., Pinkel, D., Jordan, R. C., and Albertson, D. G. (2005). Rare amplicons implicate frequent deregulation of cell fate specification pathways in oral squamous cell carcinoma. Oncogene 24,