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. 2012 Jan 23;287(11):8598–8612. doi: 10.1074/jbc.M111.322800

FIGURE 4.

FIGURE 4.

Leptin-mediated increase in GSK3β phosphorylation involves activation of Akt as well as Wnt1. A, breast cancer cells were treated with 100 ng/ml leptin for indicated time intervals. Total lysates were immunoblotted for phospho-Akt expression. The membranes were re-blotted using total Akt and actin antibodies as control. The blots are representative of multiple independent experiments. The histogram is the mean of densitometric analysis showing relative density units (RDU) of the Western blot signal for phosphorylated Akt normalized to total Akt. *, p < 0.001 compared with untreated controls. Leptin treatment increases phosphorylation of Akt. B, breast cancer cells were untreated (U) or treated with 100 ng/ml leptin alone (L), 10 μm Akt inhibitor LY294002 alone (LY), and in combination (L+LY). Total lysates were immunoblotted for phospho-GSK3β and phospho-Akt expression levels. The membranes were re-blotted using total GSK3β, total Akt, and actin antibodies as control. LY294002 reduces leptin-induced phosphorylation of GSK3β. C, breast cancer cells were transiently transfected with siAkt#1 or siAkt#2 siRNAs (Invitrogen) using FuGENE (Invitrogen). Immunoblot analysis was performed using Akt antibodies, and actin antibodies were used as control. D, breast cancer cells were transfected with siAkt#2 for 24 h followed by leptin treatment for 6 h. Total lysates were subjected to immunoblot analysis using specific antibodies for phospho-GSK3β, fibronectin, and vimentin. The membranes were re-blotted using actin. The blots are representative of multiple independent experiments. Akt silencing inhibits leptin-induced GSK3β phosphorylation, fibronectin, and vimentin expression. E, breast cancer cells were treated with 100 ng/ml leptin for indicated time intervals. Total lysates were immunoblotted using anti-Wnt1 antibodies. Immunoblots for actin levels were used as control. The blots are representative of multiple independent experiments. The histogram is the mean of densitometric analysis showing relative density units (RDU) of the Western blot signal for Wnt1 normalized to actin. *, p < 0.005 compared with untreated controls. Leptin increases Wnt1 expression. F, breast cancer cells were transiently transfected with siWnt1#1 or siWnt1#2 siRNAs (Invitrogen) using FuGENE (Invitrogen). Immunoblot analysis was performed using Wnt1 antibodies, and actin antibodies were used as control. G, breast cancer cells were transfected with siWnt1#1 for 24 h followed by leptin treatment for 6 h. Total lysates were subjected to immunoblot analysis using specific antibodies for phospho-GSK3β. The membranes were re-blotted using total GSK3β. The blots are representative of multiple independent experiments. Wnt1 inhibition inhibits leptin-induced GSK3β phosphorylation.