Supplementary Figure 3: Granulomatous aggregation in vitro is specific for TSC2-deficient macrophages.

(a) p-S6 immunofluorescence of growth factor-deprived Tsc2^fl/fl and Tsc2^fl/fl,Lyz2-Cre BMDM on day 7. Scale bar, 10 μm. (b) BMDM were deprived of growth factor overnight and re-stimulated with 10 ng/ml CSF1 or 100 ng/ml LPS for the indicated time points. Whole cell lysates were analyzed by immunoblotting. (c) Representative images of IL-4 stimulated (10 ng/ml) BMDM treated with solvent or 100 nM rapamycin for four days. Scale bar, 100 μm. (d) Cell volume of BMDM after differentiation. Shown are means ± s.e.m. (Tsc2^fl/fl n= 4, Tsc2^fl/fl,Lyz2-Cre n= 5). (e) Images of Tsc2^fl/fl,Lyz2-Cre BMDM that were treated with solvent or 100 nM rapamycin for 24 h. Scale bar, 100 μm. (f) Images of lung single cell suspensions that were cultivated in CSF1 containing media for 7 days. Treatment with solvent or 100 nM rapamycin lasted for 48h. Scale bar, 100 μm. (g) Images of Tsc2^fl/fl and Tsc2^fl/fl,Lyz2-Cre dendritic cells (DC) differentiated for 6 six days that were cultured for additional 6 days in the presence of 20 ng/ml CSF2. Scale bar, 100 μm. Right panel, whole cell lysates of Tsc2^fl/fl and Tsc2^fl/fl,Lyz2-Cre DCs were analyzed by immunoblotting with the indicated antibodies. (h) Representative images of primary human foreskin and lung fibroblasts, as well as mouse embryonic fibroblasts (MEF) transfected with Tsc2 or non-target siRNAs or left untreated as indicated. Scale bar, 200 μm. Right panel, Knockdown efficiency was confirmed by immunoblotting using the indicated antibodies. Results are representative of three (a,c,e,f), four (g), or two (b) mice per genotype or cumulative for four to five (d) mice per genotype or for 2 independent experiments (lung and foreskin) or one experiment (h).