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sc-12-0511_sm_SupplFigure1.pdf656KSupplemental Fig. 1. Expression of keratin and vimentin in ETIC tumors and cell lines. (A) Representative immunofluorescence images of four different MMTV-Wnt1 tumors analyzed for K14 (green) and K8 (red). Scale bars represent 50um (B) Quantitative analysis of K8 and K14 positive areas in individual MMTV-Wnt1 tumors. Data represent the average percent of the tumor area positive for the indicated antibody. Each bar represents the mean and SD of 9 separate images. (C) Variation of keratin staining within a single tumor. Sections of a WMG316 tumor at 100 μm intervals were analyzed for K14 (green bar) and K8 (red bar) expression as quantified by the positively stained areas. Increased representation of K8 positive cells relative to K14 is found through out the tumor. (D) WMG300 (left column) and WMG316 (right column) cells grown in monolayer, stained with the indicated antibodies against intermediate filament proteins. Antibodies are indicated as follows from top to bottom: keratin 8 (K8, red), keratin14 (K14, green), keratin 6 (K6, green), keratin 19 (K19, red), keratin 5 (K5, green) and keratin 18 (K18, red). Scale bars represent 40um. (E) Quantitative cell identification determined with use of CyteSeer image analysis is represented as the average percent of positive cells for each individual marker.
sc-12-0511_sm_SupplFigure2.tif518KSupplemental Fig. 2 Verification of keratin content assay and results of kinase inhibitor screen (A) Each spot represents the sum of the three detectable keratin populations (K8+K14+, K8+ K14- , K8- K14+) for each of the 242 kinase inhibitors. Each inhibitor was tested in three wells and the corresponding average cell count is indicated by DAPI stained nuclei. Correspondence between number of nuclei and the sum of keratin stained cells indicates efficient identification of keratin stained cells by antibody staining and image analysis. (B) Log2 fold change of cell number identified by DAPI staining relative to control wells for individual kinase inhibitors. (C) Relative fractions of K8 single positive (top panel) or K14 single positive (bottom panel) cells relative to total cell count (DAPI stain) for individual kinase inhibitor. Data represent the average of 3 replicate wells for wells with at least 200 scored cells. Inhibitor identification number is indicated on the X-axis.
sc-12-0511_sm_SupplFigure3.tif950KSupplemental Fig. 3 Investigation of ROCK and GSK3β pathways on growth and CFU. (A) Dose response relationship of R1 was investigated in the absence (solid squares) or presence of the annotated GSK3β inhibitor (inverted triangles) at the near optimal concentration of 0.3 μM. Data represent the average of 3 replicates ± SD. (B) Assessment of colony forming ability in monolayer culture with a stable ROCK 2 cell line (sh919) with increasing levels of R1 inhibitor. Data represent average of 3 replicates ± SD. ROCK2 knockdown does not alleviate stimulatory effect of R1. (C) Cytospin preparations were generated from suspension cultures of WMG300 and WMG300sh902 24hrs post-plating. Cytospins were stained for cleaved caspase-3, imaged and subsequently scored with CyteSeer analysis software. Data represent the average percent of scored cells of four separate image fields ± SD, *p-value=0.19, **p-value=0.11. (D) Cellular proliferation assay of WMG300 (left panel) and WMG300sh902 (right panel) in the presence or absence of the ROCK inhibitor (R1). Cells were plated in 96 well gelatin coated wells in triplicate in the absence of R1 (2000 cells/well) or in the presence of R1 (1 μM, 1000/cells per well). At the corresponding time point, cells were fixed, stained for K8 and K14, imaged and analyzed for DAPI positive cells via CyteSeer analysis software. Values for total cell number (DAPI) are very similar to the K8+K14+ double positive population shown.
sc-12-0511_sm_SupplFigure4.tif2306KSupplemental Fig. 4 Tumor progression of WMG300 to MTIC. (A) Tumor growth rate is indicated by the number of days necessary for tumors to reach maximum allowable size upon successive passages of WMG300T (solid triangles) and WMG49T (solid squares). Arrow indicates the tumors used for analysis in (B). (B) Representative H&E and vimentin (brown) IHC on ETIC (WMG49) and MTIC tumor (WMG300). Scale bar represents 100 μm. (C) K8 and K14 immunofluorescence of WMG300 MTIC culture. (D) FACS profile of WMG300 ETICs and WMG300 MTICs. Ethanol fixed cells were stained for K8 and K14. Live cells were stained for CD24 and CD29.
sc-12-0511_sm_SupplFigure5.tif2486KSupplemental Fig. 5 Evaluation of MLC2 phosphorylation by ROCK1. (A) Immunofluorescent analysis for vimentin (VIM) and phosphorylated MLC2 (pMLC2) of WMG300. CyteSeer software scored cells positive for VIM (red bar) and pMLC2 (green bar). Data represent the average fraction of cells positive for each antibody. Note decreased fraction of cells positive for vimentin and pMLC2 in cells treated with R1. (B) Preferential co-localization of pMLC2 in cell expressing vimentin. Cells expressing vimentin were scored for simultaneous expression of pMLC2. In the presence or absence of the R1, pMLC2 is exclusively associated with cells expressing vimentin. In the absence of R1, most vimentin positive cells also express pMLC2. In the presence of R1, a smaller fraction of the vimentin cells express pMLC2. (C) Representative images of VIM and pMLC2 stains of WMG300. The nuclei are visualized by DAPI.
sc-12-0511_sm_SupplTable1.pdf399KSupplemental Table 1 Cell count and Keratin analysis of Kinase screen.
sc-12-0511_sm_SupplTable2.pdf79KSupplemental Table 2 GeneGo Pathway analysis.
sc-12-0511_sm_SupplTable3.pdf372KSupplemental Table 3 Full details of the analysis of RNAs increased in ETICs.
sc-12-0511_sm_SupplTable4.pdf338KSupplemental Table 4 Full details of the analysis of RNAs increased in MTICs.

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