Supplementary Figure 5: TSC2-deficiency promotes CDK4-dependent proliferation in macrophages.

(a) BMDM were treated with 100 nM rapamycin or solvent control for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. (b) Freshly differentiated C57BL/6J BMDM were starved from CSF1 for 0, 4 and 12 h. Cells that were deprived from CSF1 for 12 h were then restimulated with 40 ng/ml CSF1 for 12 or 24h. Whole cell lysates of the time points were analyzed by immunoblotting. (c) C57BL/6J BMDM were stimulated with 10, 20, 30 and 40 ng/ml CSF1 for 24 h. Whole cell lysates were analyzed by immunoblotting. (d) C57BL/6J BMDM were deprived of CSF1 for 12 h. Afterwards, they were either harvested or treated with 250 nM Torin1 or solvent control, and stimulated with 20 ng/ml CSF1 for 24h. Whole cell lysates of the time points were analyzed by immunoblotting. (e) Representative IHC of CDK4 in liver sections of 3 month-old Tsc2^fl/fl and Tsc2^fl/fl,Lyz2-Cre mice. Scale bar, 100 μm. (f) Human monocyte-derived macrophages were treated with 100 nM rapamycin or 250 nM Torin in the presence of 20 ng/ml CSF1. Whole cell lysates were analyzed by immunoblotting. (g) Analysis of CSF1-induced proliferation of Tsc2^fl/fl and Tsc2^fl/fl,Lyz2-Cre BMDM treated with solvent or the indicated amounts of SC-203873. Shown are means ± s.e.m. (n= 4). (h) BMDM were treated with 1 μM PD-0332991 or solvent control and stimulated with 10 ng/ml CSF1 for 18 h. Whole cell lysates of the time points were analyzed by immunoblotting. Results are representative of two (a-f,h) or cumulative for two (g) individual experiments.