CAL-101 antagonizes CD40-CD40L–mediated CLL cell survival. (A) CD19⁺ cells from patients with CLL (n = 4) were incubated with 10μM CAL-101 and 1μg/mL CD40L for 2 hours. AKT phosphorylation at Ser473 was assessed by immunoblot. Results are shown from 1 of 4 experiments. (B) CD19⁺ cells from patients with CLL (n = 5-18) were incubated with or without various doses of CAL-101 and 1μg/mL CD40L for 48 hours. Viability was determined by annexin/PI flow cytometry and is shown relative to time-matched untreated controls. Horizontal lines represent the mean. (C) CD19⁺ cells from CLL patients (n = 3) were incubated with various concentrations of CAL-101 and 1μg/mL CD40L for 2 hours. AKT phosphorylation at Ser473 was assessed by immunoblot. Results are shown from 1 of 3 experiments. Quantification was done with the Alpha Innotech FluorChemQ MultiImage III system. (D) CD19⁺ cells from patients with CLL (n = 20) were incubated with or without 10μM CAL-101 and 800U/mL IL-4 for 48 hours. Viability was determined by annexin/PI flow cytometry and is shown relative to time-matched untreated controls. Horizontal lines represent the mean. (E) CD19⁺ cells from patients with CLL (n = 4) were incubated with or without 10μM CAL-101 (or 25μM LY294002) and 800U IL-4 for 2 hours. Western blot analysis was done to detect activation of AKT (phosphorylation at Ser473) or STAT3 (phosphorylation at Tyr705). Results are shown from 1 of 4 experiments. (F) CD19⁺ cells from patients with CLL (n = 3) were incubated with 10μM CAL-101 and 1μg/mL CD40L for 2 hours. GSK3β phosphorylation at Ser9 was assessed by immunoblot. Results are shown from 1 of 3 experiments. (G) CD19⁺ cells from patients with CLL (n = 3) were incubated with 10μM CAL-101 and 1μg/mL CD40L for 2 hours. Mcl-1 expression was assessed by immunoblot. Results are shown from 1 of 3 experiments.