Fig. 2.

Transformation of 32D cells by IL-27R requires JAK family kinase activity. 32D cells transformed to cytokine independence by IL-27R were cultured in the presence of 0.1% DMSO [● (A) and filled bars (C)] or 0.5 μM JAK inhibitor I [○ (A) and empty bars (C)] on day 0. (A) The total number of viable cells for each treatment was determined daily by trypan blue exclusion. Error bars indicate standard deviation within a representative experiment. (B) After 24 h of JAK inhibitor I treatment, the DNA content present in each phase of the cell cycle was determined by propidium iodide staining and flow cytometry. The percentage of cells that were in G₁, S, and G₂ are plotted. Error bars indicate standard deviation of triplicate samples of a representative experiment. (C) Cell viability was determined by trypan blue exclusion. Error bars indicate standard deviation within a representative experiment. (D) After 24 h of JAK inhibitor I treatment, apoptosis was determined by annexin V binding. Error bars indicate standard deviation of triplicates within a representative experiment. (E) 32D cells transformed by IL-27R were starved of serum/IL-3 for 3 h in the presence of 0.1% DMSO (lane 2), 0.5 μM (lane 3) or 2 μM (lane 4) JAK inhibitor I. Control 32D cells starved of serum/IL-3 in the presence of 0.1% DMSO for 3 h is shown in lane 1. Cell lysates were immunoblotted with antibodies that recognize the indicated proteins. Similar results were obtained with independently derived cell lines.