(A) NIH3T3 cells treated with 100 nM rapamycin, 250 nM Torin1 or vehicle for 12 hours were analyzed by immunoblotting for the phosphorylation states and levels of the indicated proteins.
(B) NIH3T3 cells treated with 100 nM rapamycin, 250 nM Torin1 or vehicle for 12 hours were processed in an immunofluorescence assay to detect lamin A (green), costained with DAPI for DNA content (blue), and imaged. Illustrative nuclei for each condition are shown.
(C) Explanation of eccentricity. The eccentricity of an elliptical object is a measure of how much the shape of the ellipse deviates from being circular. Defined mathematically, eccentricity=1−2/((a/p)+1), where a is the radius at apopasis, i.e., the farthest distance from the edge of the ellipse to its center, and p is the radius at periapsis, i.e., the closest distance to ellipse center. The eccentricity of an ellipse is necessarily between 0 and 1; a circle has an eccentricity of 0. As the eccentricity tends to 1, the ellipse becomes more elongated, and the ratio of a/p tends to infinity.
(D) Images processed in (B) were analyzed for nuclear eccentricity based on lamin A immunofluorescence by image analysis software (). Nuclear eccentricity distributions of rapamycin and Torin1 treated cells were overlaid with those from corresponding vehicle treated cells. Mean. eccentricity values for three independent fields for each condition (>500 cells total) are: DMSO (black) 0.630; rapamycin (grey) 0.628; Torin1 (red) 0.704.
(E) Immunoblot analysis of TSC2 and raptor protein levels in p53−/− MEFs with RNAi-mediated knockdown of a control protein, TSC2, or raptor.
(F) p53−/− MEFs with knockdown of TSC2, raptor, or a control protein were analyzed as in (D). Mean eccentricity values for three independent fields for each condition (>500 cells total) are: shGFP (black) 0.660; shTSC2_1 (green) 0.622; shraptor_1 (red) 0.705.
(G) Immunoblot analysis of T37/T46 4E-BP1 phosphorylation in NIH3T3 cells deprived of serum, serum and glucose, or serum and amino acids for 12 hours.
(H) NIH3T3 cells treated with 250nM Torin1 or deprived of serum, serum and glucose, or serum and amino acids for the indicated times were analyzed as in (D). The mean eccentricity value for untreated cells is 0.633 (dashed line). Error bars indicate standard error for n=3. * indicates p<0.03; # indicates p<0.01.