CD28-mediated pro-survival signaling is dependent on PI3K and Akt. (A) MM.1S cells were cultured in full serum ±10 µg/mL CD28 activating mAb (CD28.2) and ± PI3K inhibitor LY294002 or ± iAkt II at the indicated doses. Cells were collected after 2 hours assessed by western blot. (B) MM.1S cells were cultured ± serum ± 10 µg/mL CD28.2 mAb. After 24 hours, cells were isolated, permeabilized, and stained intracellularly for phosphorylated Akt, which was assessed by flow cytometry. (C) MM.1S cells were cultured ± serum ±10 µg/mL CD28 activating mAb (CD28.2), and ± PI3K inhibitor LY294002 at the indicated doses. Cells were collected after 72 hours and viability was assessed via Annexin V and 7AAD staining by flow cytometry. (D) MM.1S cells were cultured ± serum ± 10 µg/mL αCD28.2 and ± Akt inhibitor (Akt inhibitor II) at the indicated doses. Cells were collected after 72 hours and viability was assessed via Annexin V and 7AAD staining by flow cytometry. Data for panels C-D) are representative of 3 independent experiments, and data for panels A-B are representative of 2 independent experiments. *P < .05, **P < .01, ***P < .001.