![Figure 3. BCR engagement increases glycolysis in a PI-3K–dependent manner. (A) Quiescent splenic B cells were cultured in the absence (t = 0 hours) or presence of 10 μg/mL anti-Ig (♦). Anti-Ig–stimulated B cells were also cultured with 1 mM 2-DOG (2DOG) for 12 hours (▵). At 90 minutes prior to the indicated times, B-cell cultures were supplemented with [5-3H]glucose, and glycolysis was then measured as described in “Materials and methods.” The standard deviations for each time point are less than 5% of the mean of triplicate measurements, and the data are representative of 3 independent experiments. (B) Glycolysis was conducted in parallel B cells that were pretreated with 10 μM LY294002 (Ig+LY), 50 nM wortmannin (Ig+Wort), or 20 nM rapamycin (Ig+Rap) for 30 minutes and then stimulated with 10 μg/mL anti-Ig (Ig) for 18 hours. The inhibitors had no measurable affect on glycolysis in B cells cultured in medium (Med) alone (data not shown). (C) Splenic B cells from wild-type (WT) and p85α-deficient (KO) mice were cultured in the absence (Media) or presence of 10 μg/mL anti-Ig (Ig). At 90 minutes prior to the indicated times, glycolysis was measured. The standard deviations for each condition are less than 5% of the mean. (D) B cells were cultured in media alone (Med) or stimulated with 10 μg/mL anti-Ig (Ig) for 18 hours in the absence or presence of 1 mM 2-DOG. Cells were then harvested for flow cytometric analysis for size (mean fwd scatter) and total cellular protein content. Protein determinations from total cellular extracts were carried out using a Bio-Rad Protein Assay Dye Reagent according to the manufacturer's recommendations.21 (E) B cells were cultured in medium alone (Media) or stimulated with 10 μg/mL anti-Ig (Ig) for the indicated times, and the amount of lactate secreted into the tissue culture medium was then measured. The data are represented as millimoles per liter (mmol/L). In panels D-E, error bars reflect standard deviation from the mean of triplicate measurements, and the data are representative of 3 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/107/11/10.1182_blood-2005-12-4788/4/zh80110696060003.jpeg?Expires=1735514172&Signature=i3SzLa78oljfNQ2THxZi-Xfkp6gu-kh~aEq3awjnjBSbFKOVgpPf2ZO49q1FJKgn1WHq4~v10VMxvCe6dTScqLNgaWNfZwW5XXJ2dvHO0V4cUl45AQ61~AVbcjZixiTShwIHz61meTjCOr85kZM9~vESczsvZkU3wvN19oYZapdh3d-VxNiCOF81IEW-9kV-GgeuzUL~rqjIEGjau~MRSPbYLIwyNh7rNht6XKxranPWGI81M9iQcIhojNRxuGklST8UzeFcb-3LUxLXLQbjEz~n98YOpeJ7hl4vF~wVSU76g1v7qN1Rp04BbCsSTEGKII1ZwAg1V7LkIMbut6ZDwQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
BCR engagement increases glycolysis in a PI-3K–dependent manner. (A) Quiescent splenic B cells were cultured in the absence (t = 0 hours) or presence of 10 μg/mL anti-Ig (♦). Anti-Ig–stimulated B cells were also cultured with 1 mM 2-DOG (2DOG) for 12 hours (▵). At 90 minutes prior to the indicated times, B-cell cultures were supplemented with [5-3H]glucose, and glycolysis was then measured as described in “Materials and methods.” The standard deviations for each time point are less than 5% of the mean of triplicate measurements, and the data are representative of 3 independent experiments. (B) Glycolysis was conducted in parallel B cells that were pretreated with 10 μM LY294002 (Ig+LY), 50 nM wortmannin (Ig+Wort), or 20 nM rapamycin (Ig+Rap) for 30 minutes and then stimulated with 10 μg/mL anti-Ig (Ig) for 18 hours. The inhibitors had no measurable affect on glycolysis in B cells cultured in medium (Med) alone (data not shown). (C) Splenic B cells from wild-type (WT) and p85α-deficient (KO) mice were cultured in the absence (Media) or presence of 10 μg/mL anti-Ig (Ig). At 90 minutes prior to the indicated times, glycolysis was measured. The standard deviations for each condition are less than 5% of the mean. (D) B cells were cultured in media alone (Med) or stimulated with 10 μg/mL anti-Ig (Ig) for 18 hours in the absence or presence of 1 mM 2-DOG. Cells were then harvested for flow cytometric analysis for size (mean fwd scatter) and total cellular protein content. Protein determinations from total cellular extracts were carried out using a Bio-Rad Protein Assay Dye Reagent according to the manufacturer's recommendations.21 (E) B cells were cultured in medium alone (Media) or stimulated with 10 μg/mL anti-Ig (Ig) for the indicated times, and the amount of lactate secreted into the tissue culture medium was then measured. The data are represented as millimoles per liter (mmol/L). In panels D-E, error bars reflect standard deviation from the mean of triplicate measurements, and the data are representative of 3 independent experiments.
