Measurements of glycolysis and mitochondrial function are required to quantify energy metabolism in a wide variety of cellular contexts. In human pluripotent stem cells (hPSCs) and their differentiated progeny, this analysis can be challenging because of the unique cell properties, growth conditions and expense required to maintain these cell types. Here we provide protocols for analyzing energy metabolism in hPSCs and their early differentiated progenies that are generally applicable to mature cell types as well. Our approach has revealed distinct energy metabolism profiles used by hPSCs, differentiated cells, a variety of cancer cells and Rho-null cells. The protocols measure or estimate glycolysis on the basis of the extracellular acidification rate, and they measure or estimate oxidative phosphorylation on the basis of the oxygen consumption rate. Assays typically require 3 h after overnight sample preparation. Companion methods are also discussed and provided to aid researchers in developing more sophisticated experimental regimens for extended analyses of cellular bioenergetics.

The TCL1 protooncogene is overexpressed in many mature B cell lymphomas, especially from AIDS patients. To determine whether aberrant expression promotes B cell transformation, we generated a murine model in which a TCL1 transgene was overexpressed at similar levels in both B and T cells. Strikingly, transgenic mice developed Burkitt-like lymphoma (BLL) and diffuse large B cell lymphoma (DLBCL) with attendant Bcl-6 expression and mutated J H gene segments at a very high penetrance beginning at 4 months of age. In contrast, only one mouse developed a T cell malignancy at 15 months, consistent with a longer latency for transformation of T cells by TCL1. Activation of premalignant splenic B cells by means of B cell antigen receptor (BCR) engagement resulted in significantly increased proliferation and augmented AKT-dependent signaling, including increased S6 ribosomal protein phosphorylation. Transgenic spleen cells also survived longer than wild-type spleen cells in long-term culture. Together these data demonstrate that TCL1 is a powerful oncogene that, when overexpressed in both B and T cells, predominantly yields mature B cell lymphomas.T he TCL1 (T cell leukemia 1) protooncogene is expressed in CD3 Ϫ CD4 Ϫ CD8 Ϫ precursor T cells and is extinguished at the CD4 ϩ CD8 ϩ stage of thymocyte development (1). In B cells, TCL1 is first expressed in pro-B cells and remains high in naive mantle zone B cells of peripheral lymphoid tissues (1-4). Downregulation of TCL1 expression in follicle center centroblasts and centrocytes is followed by gene extinction in post-germinal center (GC) memory B cells and plasma cells (4, 5).Continued high-level TCL1 expression, because of chromosomal rearrangements, was implicated in mature peripheral T cell malignancies (6, 7). Polyclonal and oligoclonal T cell expansions preceded clonal outgrowth by many years, suggesting that additional lesions were required for transformation (8,9). Supporting this tumorigenic mechanism, transgenic mice expressing TCL1-familymember proteins exclusively in T cells developed polyclonal T cell expansions before the evolution of clonal malignancies at 15 to 20 months (10, 11). Overexpression of TCL1, or MTCP1 (mature T cell proliferation 1), in mouse T cells did not affect B cell development or produce B cell lymphomas. These findings indicate that aberrant expression of TCL1 or MTCP1 in T cells perturbs T cell homeostasis through cell autonomous pathways without inducing premalignant or malignant changes in bystander B cells.About 15% of AIDS patients develop aggressive B cell nonHodgkin lymphoma (AIDS-NHL) (12, 13). Most AIDS-NHL originate from GC or post-GC B cells, but the early events leading to AIDS-NHL remain poorly defined (13,14). Diffuse large B cell lymphoma (DLBCL) is the most prevalent type of AIDS-NHL, and these tumors generally lack consistent genetic and͞or viral tumorpromoting alterations. Recently, abundant TCL1 expression was shown in a high percentage of AIDS-NHL of post-GC origin (3,4). This discovery led us to postulate that TCL1 dy...

P rimary effusion lymphoma (PEL) is an infrequent neoplasia of severe immunodeficiency and is usually observed in those with longstanding AIDS (1, 2). PEL exhibit features both in common with and distinct from other B cell neoplasms. Like myeloma, PEL arise from postgerminal center, developmentally mature B lymphocytes (1-3). Unlike myeloma, PEL grow in body cavities as liquid effusions, are almost always infected with human herpesvirus-8 (HHV-8) and lack expression of B-lineagespecific genes (reviewed in ref.2). The loss of B lineage gene expression in PEL could be caused by DNA methylation and epigenetic silencing. In accord with this idea, ␥-herpesvirus genomes such as HHV-8, herpesvirus saimiri, and Epstein-Barr virus, are commonly methylated (4, 5). The same mechanism(s) responsible for viral DNA methylation may also be involved in methylation and silencing of B lineage-specific genes in PEL.DNA methylation in mammalian cells largely occurs on cytosines in symmetric CpG dinucleotides and is associated with repressed gene transcription (6 -10). Methyl-binding domain proteins engage methylated CpG ( m CpG) and recruit histone deacetylase (HDAC) and transcriptional repressors to form stable repression complexes that induce local chromatin remodeling and gene silencing (11-16). Early mammalian embryos and germ cells, like plants and fungi, also methylate non-CpG cytosines (17-21). A recent report indicates that peripheral blood leukocytes may methylate the internal cytosine of symmetric CCTGG but not CCAGG motifs (22). However, little is known about the gene specificity, frequency, and functional significance of this latter type of symmetric non-CpG methylation.The B29 (Ig␤͞CD79b) component of the B cell surface receptor is encoded by the B29 gene and is absent in all PEL lines and some myelomas (1, 23). The B cell-specific B29 promoter is well characterized and provides an ideal model to analyze DNA methylation in B lineage gene silencing in PEL and myeloma. Early B cell factor (EBF), in concert with Octamer, Ets, Sp1, and Ikaros transcription factors, regulates B29 promoter activity in early B cell development, whereas non-EBF factors control B29 gene expression at later stages of B cell differentiation (24). The human B29 promoter contains 20 CpG dinucleotides and 6 CCAGG or CCTGG motifs within a 450-bp span (25). CpG dinucleotides are present in single Sp1 and EBF consensusbinding sites, whereas the CCAGG and CCTGG motifs are concentrated in a central promoter control region containing essential EBF sites.Here we report that the B29 promoter in PEL and nonexpressing myeloma cells is methylated at CpG and CC(A͞T)GG sites. Because the methylation pattern observed at these B29 promoter sites is similar to that reported in epigenetic retroviral silencing, we propose that B cell gene extinction occurs through a similar mechanism. We also find that CC(A͞T)GG methylation repressed B29 promoter activity and replaced transcription factors with new protein complexes. Materials and MethodsDNA Demethylation and HDAC In...