Activation of mTORC1 and other signaling pathways in CTCL cell lines and preactivated primary cells. (A) Western blots of protein lysates from 2 spontaneously growing and 2 IL-2–dependent CTCL cell lines were performed using a set of antibodies against the listed phospho-proteins from the mTORC1, PI3K/Akt, MEK/ERK, STAT3, and STAT5 signaling pathways. Antibody against actin served as a positive control. Before cell lysis, the IL-2–dependent cell lines were cultured without IL-2 for 24 hours and stimulated afterward for 30 minutes with 100 U of the cytokine or medium alone. (B) Western blots of protein lysates from the 2 IL-2–dependent/IL-2–depleted CTCL cell lines stimulated for 30 minutes with medium alone, 100 U of IL-2, 5 ng/mL of IL-7, 20 ng/mL of IL-15, or 100 ng/mL of IL-21 were performed using the same listed antibodies as in panel A. (C) The effect of serum and nutrient depletion on mTORC1 activation. The 2 CTCL cell lines were incubated for 20 hours in RPMI medium supplemented with 10% FBS or RPMI with 1% BSA (with a subset of samples cultured for the last 2 hours in PBS with 1% BSA), lysed, and analyzed for phosphorylation status of the mTORC1, PI3K, and MEK1/2 target proteins using the depicted antibodies. The 1% BSA/PBS cultures restimulated for 1 hour with the complete 10% FBS/RPMI medium served as positive controls. (D) Western blots of protein lysates from blood-circulating lymphoma cells isolated from 3 patients CTCL with an advanced leukemic (Sézary) phase and stimulated for 30 minutes with medium alone, 100 U of IL-, or 100 ng/mL of IL-21. Whereas some of the cell populations were examined directly after isolation (left), other were preactivated by a 7-day culture with a mitogen PHA (right).