Revealed: Reasons PTEN Makes Everyone Much Happier

01, t test). J/EGFP-positive sphere cells also formed more and larger colonies in soft agar than J/EGFP-negative cells ( Figure?3F, p PTEN cells reformed more spheres in single cell dilution assays additionally suggesting increased self-renewal capacity ( Figure?3G, p = 0.013, Fisher's exact test). Single J/EGFP-negative cells died or did not form spheres. Together with the expression studies in melanoma patient specimens and cell lines (Figure?1 and Figure?S1), our data suggest click here a JARID1B-expressing subpopulation that remains in?a slow-cycling state, but when released from its microenvironment, it can give rise to a rapidly proliferating progeny that reconstitutes the parental heterogeneity of JARID1B-positive and -negative cells. Particularly when the culture conditions supported the survival of cells with inherent self-renewal potential (hESCM4), single-seeded J/EGFP-positive cells were superior to single J/EGFP-negative cells regarding their capacity to repopulate. Titrated xenotransplantation assays were done in NOD/LtSscidIL2R��null mice according to the improved protocol published recently ( Quintana et?al., 2008). We subcutaneously injected 100, 10, or 1 cell from the FACS-isolated J/EGFP-positive versus?-negative subpopulation cultured in conventional medium (each n = 20, Figure?3H). Unsorted cells were injected as controls. The absolute tumor initiation rate of J/EGFP-positive and -negative cells was nearly identical in line with the observations made by Quintana et?al. We found in all titration steps that J/EGFP-negative cells started to grow earlier. Although not a statistically significant difference, this is consistent with our in?vitro observations of colony formation TSA HDAC nmr assays. Given the paradox of an increased in?vitro self-renewal capacity without any effect on in?vivo tumor initiation, we asked whether JARID1B could still be important for the continuous growth of melanomas. JARID1B may not be required for tumor initiation but in the maintenance of established tumors thus reflecting two different biological processes. To address this, JARID1B was stably knocked down in WM3734, WM35, and WM3899 melanoma cells (Figure?4) and in primary foreskin melanocytes (FOM) as control (Figures S4H�CS4J). JARID1B knockdown efficiency was validated as summarized in Figure?S4A�CF. Two shRNA clones targeting different mRNA regions of JARID1B were selected for analysis. Off target effects were excluded by computerized analysis (http://rnai.cs.unm.edu/offTarget).