Figure 4.

Reduced anabolic metabolism in M-CSF–stimulated Rptor^−/− myeloid cells. (A) Immunoblot analysis of phosphorylated and total proteins of S6, 4E-BP1, ERK1/2, and β-actin in fresh (0 h) or M-CSF–stimulated (10 ng/ml, for the indicated times) Lin^– cells from WT and Rptor^−/− mice. (B) 2-NBDG staining of CD115⁺ and CD115^– cells in WT mice. (C) 2-NBDG staining of CD11b⁺ cells from the spleen of WT and Rptor^−/− mice, with mean fluorescence intensity (MFI) plotted within graph. (D) 2-NBDG staining of CD11b⁺F4/80⁺ cells from Lin^– cells of WT and Rptor^−/− mice stimulated with M-CSF (10 ng/ml) for 3 d, with MFI plotted within graph. (E) Measurement of ECAR in developing myeloid cells (stimulated with 10 ng/ml M-CSF for 2 d) in response to the indicated mitochondrial inhibitors. Oligo, Oligomycin; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. (F) Measurement of OCR in Lin^– cells from WT and Rptor^−/− mice stimulated with M-CSF (10 ng/ml) for 2 d. (G) Cell size of CD11b⁺F4/80⁺ cells from WT and Rptor^−/− mice, with MFI plotted above graph. (H) Representative images (left) and quantification of mitochondrial content per cell (right) of Lin^– cells from WT and Rptor^−/− mice stimulated with M-CSF (10 ng/ml) for 2 d. Data are mean ± SEM and representative of two (A and B), six (D), three (C and F), or four (E, G, and H) independent experiments. ***, P < 0.001; Student’s t test.