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Additional Supporting Information may be found in the online version of this article.

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STEM_538_sm_suppinfofig1.tif242KSupplementary Figure 1. Serial dilution experiment of colony and sphere forming activity. Wt and Pten/TP53 null primary prostate epithelial cells were plated in the absence (A) or presence (B) of MatrigelTM in a dilution series ranging from 0.1 to 1 × 104 cells per well. Graphs show the number of colonies or spheres grown at each dilution plotted versus the input cell number. Trend lines (red lines) that best approximate the curves were obtained by linear regression analysis. R2 is the coefficient of determination. The data are reported as mean ± SD.
STEM_538_sm_suppinfofig2.tif1795KSupplementary Figure 2. Immunophenotype of the differentiated luminal and neuroendocrine cells within wt and Pten/TP53 null protospheres and prostate epithelial colonies. (A) Immunofluorescent images of wt and Pten-/-TP53-/- prostate epithelial colonies stained for chromogranin A. (B) Immunofluorescent images of cytospin preparations of wt and Pten-/-TP53-/- cells isolated from spheres and stained for chromogranin A. (B) Quantification of the percentage of chromogranin A positive cells assayed in B. (C) Immunofluorescent images of cytospin preparations of wt and Pten-/-TP53-/- cells isolated from spheres and stained for mouse prostatic secretory proteins (mPSP). (C) Quantification of the percentage of mPSP positive cells assayed in C.
STEM_538_sm_suppinfofig3.tif547KSupplementary Figure 3. Wt and Pten-/-TP53-/- LSC cells are enriched for colony and sphere forming activity in vitro FACS plots in (A) and (B) show gates drawn for sorting of LSC (Lin–Sca-1highCD49f high) [Red], Sca (Lin–Sca-1+CD49f lo/–) [Blue], and CD49f (Lin–Sca-1–CD49f+) [Green] subpopulations. Primary prostate cells from wt animals were sorted into the populations shown in A and those from Pten-/-TP53-/- animals were sorted into the populations shown in B. Each population was plated in triplicates at a density of 10,000 cells per well with or without MatrigelTM. Bar graphs in (C) and (D) show the number of wt and Pten-/-TP53-/- colonies, respectively, that grew out 9 days later. Bar graphs in (E) and (F) show the number of wt and Pten-/-TP53-/-spheres, respectively, that grew out 15 days later.
STEM_538_sm_suppinfofig4.tif341KSupplementary Figure 4. A dose dependent effect of mTOR and AR inhibitors on colony and sphere forming activity of wt and Pten-/-TP53-/- null prostate epithelial cells. Wt and Pten/TP53 null primary prostate epithelial stem/progenitor cells were enumerated for CFU (A) and SFU (B) in the absence or presence of different concentrations of drugs targeting the AKT/mTORC1 pathway (Rapamycin and Triciribine) and AR pathway (Bicalutamide).

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